Transcription factor - E2F-5

ABSTRACT

Two novel transcription factors belonging to the E2F gene family, are disclosed. These are human and murine E2F-5. They can interact with DP-1 and p130.

[0001] This invention relates to a novel transcription factor and to itsproduction and uses.

[0002] The molecular events that occur during the cell cycle need to beintegrated with the transcription apparatus so that gene expression canbe synchronised with cell cycle progression.

[0003] Recently, a transcription factor called E2F (or DRTF1) has beenidentified and shown to bind to pRb, the protein product of theretinoblastoma susceptibility gene, an anti-oncogene or tumoursuppressor gene (see for example Wagner and Green, Nature 352, 189-190,1991). It is widely believed that the cellular transcription factor E2Ffunctions as a key component in cell cycle control because it associateswith important cell cycle regulating proteins, such as theretinoblastoma gene product (pRb), p107, cyclins and cyclin-dependentkinases, and furthermore its transcriptional activity is modulated bycertain viral oncoproteins, such as adenovirus Ela, SV40 large Tantigen, and the human papilloma virus E7 protein.

[0004] It is believed that the transcription factor E2F (or DRTF1) playsan important role in integrating cell cycle events with thetranscription apparatus because, during cell cycle progression inmammalian cells, it undergoes a series of periodic interactions withmolecules that are known to be important regulators of cellularproliferation. For example, the retinoblastoma tumour suppressor geneproduct (pRb), which negatively regulates progression from G1 into Sphase, and is frequently modified in tumour cells, binds to E2F.Similarly, the pRb-related protein p107 occurs predominantly in an Sphase complex with E2F. Both pRb and p107 repress the transcriptionalactivity of E2F, which is likely to be fundamentally important forregulating cellular proliferation because E2F binding sites occur in thecontrol regions of a variety of genes that are involved withproliferation, such as c-myc and p34^(cdc2). Furthermore, mutant Rbproteins, encoded by alleles isolated from tumour cells, fail to bind toE2F, and hence are unable to interfere with E2F site-dependenttranscriptional activation. Another important feature of E2F is thatcertain viral oncoproteins, such as adenovirus Ela, SV40 large T antigenand human papilloma virus E7, modulate its activity by sequestering pRband p107 from the inactive transcription factor. This effect requiresregions in these viral proteins that are necessary for transformation oftissue culture cells and hence to overcome growth control. Thus, theability of these oncoproteins to regulate E2F may be the means by whichthey over-ride the normal mechanisms of cellular growth control and,conversely, transcriptional repression by pRb may be the basis ofpRb-mediated negative growth control.

[0005] A potential mechanism for integrating thetranscription-regulating properties of pRb and p107 with other cellcycle events was suggested by the identification of cyclin A and thecdc2-related cyclin-dependent kinase p33^(cdk2) in the E2F complex.Cyclin A is necessary for progression through S phase, a function thatcould perhaps be mediated through its ability to recruit thecyclin-dependent kinase p33^(cdk2) to E2F. Taken together these datasuggest that E2F is a transcription factor whose primary role may be torelay cell cycle events to the transcription apparatus via moleculessuch a pRb, p107, cyclins and cdks, thus ensuring that gene expressionis synchronised and integrated with cell cycle progression.

[0006] More recently, a transcription factor with the properties of E2Fhas been cloned and sequenced (Helin et al, Cell 70 (1992), 337-350 andKaelin et al, Cell 70 (1992), 351-364).

[0007] We have now surprisingly found a further two new proteins whichappear to be new members of the E2F gene family, which we have calledE2F-5. The cDNA sequence of human E2F-5 is presented in FIG. 1A, as isthe amino acid sequence of this protein. The corresponding sequences formurine E2F-5 appear in FIG. 9A. These new proteins are referred to asE2F-5 and this nomenclature will be used in this specification.

[0008] It has been found that E2F-5 is one of a family of relatedtranscription factor components. Members of this family are believed tointeract with DP proteins to form a series of transcription factors. DPproteins (or polypeptides) include DP-1, DP-2 and DP-3 although thefirst of these will usually be contemplated in preference to the others.

[0009] The invention in a first aspect provides a protein as shown inFIG. 1A or 9A, homologues thereof, and fragments of the sequence andtheir homologues, which can function as a mammalian transcriptionfactor. In particular, the invention provides a polypeptide (preferablyin substantially isolated form) comprising:

[0010] (a) E2F-5;

[0011] (b) the protein of FIG. 1A or 9A;

[0012] (c) a mutant, allelic variant or species homologue of (a) or (b);

[0013] (d) a protein at least 70% homologous to (a) or (b);

[0014] (e) a fragment of any one of (a) to (d) capable of forming acomplex with a DP protein, pRb, p107 and/or p130; or

[0015] (f) a fragment of any of (a) to (e) of at least 15 amino acidslong.

[0016] All polypeptides within this definition are referred to below aspolypeptide(s) according to the invention.

[0017] The proteins pRb, p107, DP proteins and p130 are referred toherein as complexing proteins or “complexors” as they may form a complexwith the proteins of the invention. Under certain conditions E2F-5 mayonly bind weakly to pRb.

[0018] A polypeptide of the invention will be in substantially isolatedform if it is in a form in which it is free of other polypeptides withwhich it may be associated in its natural environment (eg the body). Itwill be understood that the polypeptide may be mixed with carriers ordiluents which will not interfere with the intended purpose of thepolypeptide and yet still be regarded as substantially isolated.

[0019] The polypeptide of the invention may also be in a substantiallypurified form, in which case it will generally comprise the polypeptidein a preparation in which more than 90%, eg. 95%, 98% or 99% of thepolypeptide in the preparation is a polypeptide of the invention.

[0020] Mutant polypeptides will possess one or more mutations which areadditions, deletions, or substitutions of amino acid residues.Preferably the mutations will not affect, or substantially affect, thestructure and/or function and/or properties of the polypeptide. Thus,mutants will suitably possess the ability to be able to complex with DPproteins, pRb, p107 and/or p130. Mutants can either be naturallyoccurring (that is to say, purified or isolated from a natural source)or synthetic (for example, by performing site-directed mutagenesis onthe encoding DNA). It will thus be apparent that polypeptides of theinvention can be either naturally occurring or recombinant (that is tosay prepared using genetic engineering techniques).

[0021] An allelic variant will be a variant which will occur naturallyin a human or in an, eg. murine, animal and which will function toregulate gene expression in a substantially similar manner to theprotein in FIG. 1A or 9A.

[0022] Similarly, a species homologue of the protein will be theequivalent protein which occurs naturally in another species, and whichperforms the equivalent function in that species to the protein of FIG.1A or 9A. Within any one species, a homologue may exist as severalallelic variants, and these will all be considered homologues of theprotein. Allelic variants and species homologues can be obtained byfollowing the procedures described herein for the production of theprotein of FIG. 1A or 9A and performing such procedures on a suitablecell source, eg from human or a rodent, carrying an allelic variant oranother species. Since the protein may be evolutionarily conserved itwill also be possible to use a polynucleotide of the invention to probelibraries made from human, rodent or other cells in order to obtainclones encoding the allelic or species variants. The clones can bemanipulated by conventional techniques to identify a polypeptide of theinvention which can then be produced by recombinant or synthetictechniques known per se. Preferred species homologues include mammalianor amphibian species homologues.

[0023] A protein at least 70% homologous to that in FIG. 1A or 9A isincluded in the invention, as are proteins at least 80 or 90% and morepreferably at least 95% homologous to the protein shown in theseFigures. This will generally be over a region of at least 20, preferablyat least 30, for instance at least 40, 60 or 100 or more contiguousamino acids. Methods of measuring protein homology are well known in theart and it will be understood by those of skill in the art that in thepresent context. Homology is usually calculated on the basis of aminoacid identity (sometimes referred to as “hard homology”).

[0024] Generally, fragments of the polypeptide in FIG. 1A or 9A or itsallelic variants or species omologues thereof capable of forming acomplex with the complexors will be at least 10, preferably at least 15,for example at least 20, 25, 30, 40, 50 or 60 amino acids in length.

[0025] It will be possible to determine whether fragments form a complexwith the complex of proteins by providing the complexor protein and thefragment under conditions in which they normally form a trans-activatingtranscription factor, and determining whether or not a complex hasformed. The determination may be made by, for example, measuring theability of the complex to bind an E2F binding site in vitro, oralternatively determining the molecular weight of the putative complexby methods such as SDS-PAGE.

[0026] Preferred fragments include those which are capable of forming atrans-activation complex with DP-1 or other complexors. The examplesherein describe a number of methods to analyse the function of theprotein and these may be adapted to assess whether or not a polypeptideis capable of forming a trans-activation complex with the DP-1 protein.For example, the polypeptide can be added to the complexor in thepresence of a reporter gene construct adapted to be activated by theDP-1/E2F-5 complex (for example, see FIG. 10 of WO-A-94/10307 in thename of the Medical Research Council). Such an experiment can determinewhether the polypeptide fragment has the necessary activity.

[0027] A polypeptide of the invention may be labelled with a revealingor detectable label. The (revealing) label may be any suitable labelwhich allows the polypeptide to be detected. Suitable labels includeradioisotopes, e.g. ¹²⁵I, enzymes, antibodies and linkers such asbiotin. Labelled polypeptides of the invention may be used in diagnosticprocedures such as immunoassays in order to determine the amount ofE2F-5 protein in a sample.

[0028] A polypeptide or labelled polypeptide according to the inventionmay also be fixed to a solid phase, for example the wall of animmunoassay dish.

[0029] A second aspect of the invention relates to a polynucleotidewhich comprises:

[0030] (a) a sequence of nucleotides shown in FIG. 1A or 9A;

[0031] (b) a sequence complementary to (a);

[0032] (c) a sequence capable of selectively hybridising to a sequencein either (a) or (b);

[0033] (d) a sequence encoding a polypeptide as defined in the firstaspect; or

[0034] (e) a fragment of any of the sequences in (a) to (d).

[0035] The present invention thus provides a polynucleotide, suitably insubstantially isolated or purified form, which comprises a contiguoussequence of nucleotides which is capable of selectively hybridizing tothe sequence of FIG. 1A or 9A or to a complementary sequence.

[0036] Polynucleotides of the invention include a DNA sequence in FIG.1A or 9A and fragments thereof capable of selectively hybridizing to thesequence of FIG. 1A or 9A. A further embodiment of the inventionprovides a DNA coding for the protein in FIG. 1A or 9A or a fragmentthereof.

[0037] The polynucleotide may also comprise RNA. It may also be apolynucleotide which includes within it synthetic or modifiednucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothionate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes of thepresent invention, it is to be understood that the oligonucleotidesdescribed herein may be modified by any method available in the art.Such modifications may be carried out in order to enhance the in vivoactivity or lifespan of oligonucleotides of the invention used inmethods of therapy.

[0038] A polynucleotide capable of selectively hybridizing to the DNA ofFIG. 1A or 9A will be generally at least 70%, preferably at least 80 or90% and optimally at least 95% homologous to the DNA of FIG. 1A or 9Aover a region of at least 20, preferably at least 30, for instance atleast 40, 60 or 100 or more contiguous nucleotides. Thesepolynucleotides are within the invention.

[0039] A polynucleotide of the invention will be in substantiallyisolated form if it is in a form in which it is free of otherpolynucleotides with which it may be associated in its naturalenvironment (usually the body). It will be understood that thepolynucleotide may be mixed with carriers or diluents which will notinterfere with the intended purpose of the polynucleotide and it maystill be regarded as substantially isolated.

[0040] A polynucleotide according to the invention may be used toproduce a primer, e.g. a PCR primer, a probe e.g. labelled with arevealing or detectable label by conventional means using radioactive ornon-radioactive labels, or the polynucleotide may be cloned into avector. Such primers, probes and other fragments of the DNA of FIG. 1Aor 9A will be at least 15, preferably at least 20, for example at least25, 30 or 40 nucleotides in length, and are also encompassed within theinvention.

[0041] Polynucleotides, such as a DNA polynucleotides according to theinvention may be produced recombinantly, synthetically, or by any meansavailable to those of skill in the art. It may be also cloned byreference to the techniques disclosed herein.

[0042] The invention includes a double stranded polynucleotidecomprising a polynucleotide according to the invention and itscomplement.

[0043] A third aspect of the invention relates to an (eg. expression)vector suitable for the replication and expression of a polynucleotide,in particular a DNA or RNA polynucleotide, according to the invention.The vectors may be, for example, plasmid, virus or phage vectorsprovided with an origin of replication, optionally a promoter for theexpression of the polynucleotide and optionally a regulator of thepromoter. The vector may contain one or more selectable marker genes,for example an ampicillin resistance gene in the case of a bacterialplasmid or a neomycin resistance gene for a mammalian vector. The vectormay be used in vitro, for example for the production of RNA or used totransfect or transform a host cell. The vector may also be adapted to beused in vivo, for example in a method of gene therapy.

[0044] Vectors of the third aspect are preferably recombinant replicablevectors. The vector may thus be used to replicate the DNA. Preferably,the DNA in the vector is operably linked to a control sequence which iscapable of providing for the expression of the coding sequence by a hostcell. The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences. Such vectors may be transformed or transfected into asuitable host cell to provide for expression of a polypeptide of theinvention.

[0045] A fourth aspect of the invention thus relates to host cellstransformed or transfected with the vectors of the third aspect. Thismay allow for the replication and expression of a polynucleotideaccording to the invention, including the sequence of FIG. 1A or 9A orthe open reading frame thereof. The cells will be chosen to becompatible with the vector and may for example be bacterial, yeast,insect or mammalian.

[0046] A polynucleotide according to the invention may also be insertedinto the vectors described above in an antisense orientation in order toprovide for the production of antisense RNA. Antisense RNA or otherantisense polynucleotides may also be produced by synthetic means. Suchantisense polynucleotides may be used in a method of controlling thelevels of the E2F-5 protein in a cell. Such a method may includeintroducing into the cell the antisense polynucleotide in an amounteffective to inhibit or reduce the level of translation of the E2F-5mRNA into protein. The cell may be a cell which is proliferating in anuncontrolled manner such as a tumour cell.

[0047] Thus, in a fifth aspect the invention provides a process forpreparing a polypeptide according to the invention which comprisescultivating a host cell transformed or transfected with an (expression)vector of the third aspect under conditions providing for expression (bythe vector) of a coding sequence encoding the polypeptide, andrecovering the expressed polypeptide.

[0048] The invention in a sixth aspect also provides (monoclonal orpolyclonal) antibodies specific for a polypeptide according to theinvention. Antibodies of the invention include fragments, thereof aswell as mutants that retain the antibody's binding activity. Theinvention further provides a process for the production of monoclonal orpolyclonal antibodies to a polypeptide of the invention. Monoclonalantibodies may be prepared by conventional hybridoma technology usingthe proteins or peptide fragments thereof as an immunogen. Polyclonalantibodies may also be prepared by conventional means which compriseinoculating a host animal, for example a rat or a rabbit, with apolypeptide of the invention and recovering immune serum.

[0049] Fragments of monoclonal antibodies which can retain their antigenbinding activity, such Fv, F(ab′) and F(ab₂)′ fragments are included inthis aspect of the invention. In addition, monoclonal antibodiesaccording to the invention may be analyzed (eg. by DNA sequence analysisof the genes expressing such antibodies) and humanized antibody withcomplementarity determining regions of an antibody according to theinvention may be made, for example in accordance with the methodsdisclosed in EP-A-0239400 (Winter).

[0050] The present invention further provides compositions comprisingthe antibody or fragment thereof of the invention together with acarrier or diluent. Such compositions include pharmaceuticalcompositions in which case the carrier or diluent will bepharmaceutically acceptable.

[0051] Polypeptides of the invention can be present in compositionstogether with a carrier or diluent. These compositions includepharmaceutical compositions where the carrier or diluent will bepharmaceutically acceptable.

[0052] Pharmaceutically acceptable carriers or diluents include thoseused in formulations suitable for oral, rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intrathecal andepidural) administration. The formulations may conveniently be presentedin unit dosage form and may be prepared by any of the methods well knownin the art of pharmacy. Such methods include the step of bringing intoassociation the active ingredient with the carrier which constitutes oneor more accessory ingredients. In general the formulations are preparedby uniformly and intimately bringing into association the activeingredient with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product.

[0053] For example, formulations suitable for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants buffers, bacteriostats and solutes which renderthe formulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the polypeptide to blood componentsor one or more organs.

[0054] Polypeptides according to the invention, antibodies or fragmentsthereof to polypeptides according to the invention and theabove-mentioned compositions may be used for the treatment, regulationor diagnosis of conditions, including proliferative diseases, in amammal including man. Such conditions include those associated withabnormal (eg at an unusually high or low level) and/or aberrant (eg dueto a mutation in the gene sequence) expression of one or moretranscription factors such as the DP or E2F proteins or related familymembers. The conditions also include those which are brought about byabnormal expression of a gene whose gene product is regulated by theprotein of FIG. 1A or 9A. Treatment or regulation of conditions with theabove-mentioned peptides, antibodies, fragments thereof and compositionsetc. will usually involve administering to a recipient in need of suchtreatment an effective amount of a polypeptide, antibody, fragmentthereof or composition, as appropriate.

[0055] The invention also provides antibodies, and fragments thereof,targeted to this region in order to inhibit the activation oftranscription factors via the disruption of the formation of theE2F-5-DP protein complex.

[0056] The present invention further provides a method of performing animmunoassay for detecting the presence or absence of a polypeptide ofthe invention in a sample, the method comprising:

[0057] (a) providing an antibody according to the invention;

[0058] (b) incubating the sample with the antibody under conditions thatallow for the formation of an antibody-antigen complex; and

[0059] (c) detecting, if present, the antibody-antigen complex.

[0060] In another aspect, the invention provides a novel assay foridentifying putative chemotherapeutic agents for the treatment ofproliferative or viral disease which comprises bringing into contact aDP protein or a derivative thereof, a polypeptide of the invention and aputative chemotherapeutic agent, and measuring the degree of inhibitionof formation of the DP/E2F-5 protein complex caused by the agent. It maynot be necessary to use complete DP-1 and/or E2F-5 protein in the assay,as long as sufficient of each protein is provided such that under theconditions of the assay in the absence of agent, they form aheterodimer.

[0061] The cloning and sequencing of DP-1 (and E2F 1,2 and 3) are knownin the art and methods for the recombinant expression and preparation ofantibodies to DP-1 can be found in WO-A-94/10307.

[0062] Thus, the invention provides a screening method for identifyingputative chemotherapeutic agents for the treatment of proliferativedisease which comprises:

[0063] (A) bringing into contact:

[0064] (i) a DP polypeptide;

[0065] (ii) a polypeptide of the first aspect, and

[0066] (iii) a putative chemotherapeutic agent;

[0067] under conditions in which the components (i) and (ii) in theabsence of (iii) form a complex; and

[0068] (B) measuring the extent to which component (iii) is able todisrupt said complex.

[0069] In the assay, any one or more of the three components may belabelled, eg with a radioactive or calorimetric label, to allowmeasurement of the result of the assay. Putative chemotherapeutic agentsinclude peptides of the invention.

[0070] Variants, homologues and fragments of DP proteins are defined ina corresponding manner to the variants, homologues and fragments of theE2F-5 protein.

[0071] The complex of (i) and (ii) may be measured, for example, by itsability to bind an E2F DNA binding site in vitro. Alternatively, theassay may be an in vivo assay in which the ability of the complex toactivate a promoter comprising an E2F binding site linked to a reportergene is measured. The in vivo assay may be performed for example byreference to the examples which show such an assay in yeast, insect,amphibian or mammalian cells.

[0072] Candidate therapeutic agents which may be measured by the assayinclude not only polypeptides of the first aspect, but in particularfragments of 10 or more amino acids of:

[0073] (a) the protein of FIG. 1A or 9A;

[0074] (b) an allelic variant or species homologue thereof; or

[0075] (c) a protein at least 70% homologous to (a).

[0076] Vectors carrying a polynucleotide according to the invention or anucleic acid encoding a polypeptide according to the invention may beused in a method of gene therapy. Such gene therapy may be used to treatuncontrolled proliferation of cells, for example a tumour cell. Methodsof gene therapy include delivering to a cell in a patient in need oftreatment an effective amount of a vector capable of expressing in thecell either an antisense polynucleotide of the invention in order toinhibit or reduce the translation of E2F-5 mRNA into E2F-5 protein or apolypeptide which interferes with the binding of E2F-5 to a DP proteinor a related family member.

[0077] The vector is suitably a viral vector. The viral vector may beany suitable vector available in the art for targeting tumour cells. Forexample, Huber et al (Proc. Natl. Acac. Sci. USA (1991) 88, 8039) reportthe use of amphotrophic retroviruses for the transformation of hepatoma,breast, colon or skin cells. Culver et al (Science (1992) 256;1550-1552) also describe the use of retroviral vectors in virus-directedenzyme prodrug therapy, as do Ram et al (Cancer Research (1993) 53;83-88). Englehardt et al (Nature Genetics (1993) 4; 27-34 describe theuse of adenovirus based vectors in the delivery of the cystic fibrosistransmembrane conductance product (CFTR) into cells.

[0078] The invention contemplates a number of assays. Broadly, these canbe classified as follows.

[0079] 1. Conducting an assay to find an inhibitor of E2F-5trans-activation (that is to say, inhibition of activation oftranscription). This inhibitor may therefore inhibit binding of E2F-5 toDNA (usually at the E2F binding site). Potentially suitable inhibitorsare proteins, and may have a similar or same effect as p107. Thussuitable inhibitory molecules may comprise fragments, mutants, allelicvariants, or species homologues of p107 in the same manner as definedfor proteins of the first aspect.

[0080] 2. Assaying for inhibitors of (hetero)dimerisation. Suchinhibitors may prevent dimerisation of E2F-5 (or a polypeptide of thefirst aspect) with a complexor, for example a DP protein, such as DP-1.Of course the inhibitor can be a fragment, mutant, allelic variant orspecies homologue of a DP protein in a similar manner as defined for theproteins of the first aspect.

[0081] 3. A third category of assay is to find inhibitors ofphosphorylation. It is thought that E2F-5 (and other proteins of thefirst aspect) might be activated by phosphorylation. Therefore, aninhibitor of phosphorylation is likely to inhibit E2F-5 trans-activationproperties (and may therefore, ultimately have the same effect as theinhibitors found in either of the two previous assays). Phosphorylationis by cdk's and so an inhibitor of this phosphorylation is one that iscontemplated by such assays.

[0082] The invention contemplates a number of therapeutic uses. Forexample, gene therapy using a nucleic acid a sequence that is antisenseto E2F-5. Molecules that can bind to a DP-1 protein and thereby form aninactive complex with the DP protein are additionally contemplated.Suitable molecules include those of the first aspect apart from E2F-5itself. Such molecules may be mutants of E2F-5, and are often referredto as dominant negative molecules in the art.

[0083] The invention contemplates the treatment or prophylaxis ofdiseases that are based on the uncontrolled proliferation of cells, orwhere uncontrolled proliferation is an important or essentialpathological aspect of the disease. This includes cancer, viral disease,self proliferation itself as well as auto immune diseases suchpsoriasis. One may also wish to temporarily inhibit the growth ofdividing cells, for example hematopoieric stem cells and/or bone marrowcells. In these aspects one is generally seeking to prevent, inhibit orinterfere with the activity of E2F-5.

[0084] In contrast some diseases and conditions can be treated byincreasing E2F-5 expression, for example by promoting or inducingoverexpression. This preferably results in apoptosis, sometimes known asprogrammed cell death. Overexpression of the E2F-5 protein can result indeath of the cell, and therefore this aspect can also be used in thetreatment of cancer. One aim is therefore to increase the activity ofE2F-5. Similar uses are known for E2F-1 (Qin et al, PNAS USA 91 (inpress)).

[0085] It should be borne in mind that the E2F-5 gene might be mutatedin tumour cells. In that event, the mutated gene could be used indiagnosis of a condition resulting from the mutation. It also lendsitself to treatment via the mutated gene.

[0086] The following two Examples describe the isolation andcharacterization of the novel protein and DNA of the invention fromhuman and murine sources, respectively. However, other e.g. mammaliansources are within the scope of the present invention and othermammalian homologues of the protein may be isolated in an analogousmanner. The Examples are presented here by way of illustration and arenot to be construed as limiting on the invention.

EXAMPLE 1 SUMMARY

[0087] E2F DNA binding sites are found in a number of genes whoseexpression is tightly regulated during the cell cycle. The activity ofE2F transcription factors is regulated by association with specificrepressor molecules that can bind and inhibit the E2F transactivationdomain. For E2F-1, 2 and 3 the repressor is the product of theretinoblastoma gene, pRb. E2F-4 interacts with pRb-related p107 and notwith pRb itself. Recently, a cDNA encoding a third member of theretinoblastoma gene family, p130, was isolated. p130 also interacts withE2F DNA binding activity, primarily in the G₀ phase of cell cycle. Wereport here the cloning of a fifth member of the E2F gene family. Thehuman E2F-5 cDNA encodes a 346-amino acid protein with a predictedmolecular mass of 38 kDa. E2F-5 is more closely related to E2F-4 (78%similarity) than to E2F-1 (57% similarity). E2F-5 resembles the otherE2Fs in that it binds to a consensus E2F site in a cooperative fashionwith DP-1. Using a specific E2F-5 antiserum, we show that underphysiological conditions E2F-5 interacts preferentially with p130.

Introduction.

[0088] E2F is the name given to a group of heterodimeric transcriptionfactors that are composed of an E2F-like and a DP-like subunit [27]. E2FDNA binding sites are present in the promoters of a number of geneswhose expression is regulated during the cell cycle and evidence existsto indicate that the presence of these E2F sites contributes to the cellcycle-regulated expression of these genes [13, 28, 38].

[0089] E2F DNA binding activity has been found in complex with theretinoblastoma protein (pRb) and the pRb-related p107 and p130 [6, 10,29, 37]. This group of proteins shares a conserved motif, the “pocket”,that is involved in binding to both cellular and viral proteins. Forthis reason, the group of pRb-like proteins is collectively known as thepocket protein family. Complexes between E2F and the various pocketproteins are likely to have different functions in cell cycle regulationas their appearance differs during the cell cycle. E2F in complex withpRb is found mostly in G₁ phase of the cell cycle [5-7, 11]. Incontrast, complexes between p107 and E2F persist during the cell cycle,but their composition is variable. In G₁, apart from E2F and p107,cyclin E and cdk2 are present. In S phase, cyclin E is replaced bycyclin A in the E2F/p107 complex [29, 37]. The functional significanceof the presence of these cyclin/cdk complexes in the p107/E2F complex isnot clear at present. In quiescent cells, a complex between E2F and p130is the most prominent E2F DNA binding species. This complex disappearsas cells emerge from quiescence, suggesting a role for p130-interactingE2F activity in cell cycle entry [10].

[0090] The ability of E2F to activate transcription is regulated bycomplex formation with the pocket proteins. Complex formation betweenE2F and pRb is subject to regulation by phosphorylation. Only thehypophosphorylated species of pRb interact with E2F, indicating that thephosphorylation of pRb by cyclin/cdk complexes controls the interactionbetween E2F and pRb during the cell cycle [5-7, 11].

[0091] The crucial role of E2F transcription factors in cell cycleregulation is emphasized by the finding that enforced expression of E2FDNA binding activity causes cells to progress from G₁ into S and G₂/Mphases of the cell cycle [3] and E2F can stimulate quiescent cells toinitiate DNA synthesis [23]. Importantly, over-expression of E2F,together with an activated ras oncogene can cause oncogenictransformation of primary rodent fibroblasts [3].

[0092] To date four different E2F-like polypeptides have been isolated.E2F-1, 2 and 3 are found only in complex with pRb, whereas E2F-4interacts preferentially with p107 [3, 15. 19, 22, 24, 30, 36]. Howcomplex formation between E2F and p107 and E2F and p130 is regulated iscurrently not known. To begin to address the regulation of the E2F/p107complex and the E2F/p130 complex, we have searched for additionalmembers of the E2F gene family. We report here the cloning of a fifthmember of the E2F gene family that interacts preferentially with p130.

Materials and Methods.

[0093] Yeast two-hybrid screen

[0094] Yeast strain Y190 [17], containing the ‘bait’ plasmid pPC97-p107,was transformed with a day 14.5 CD1 mouse embryo library [8] using thelithium acetate method [34]. Two million transformants were selected forgrowth on plates lacking histidine and supplemented with 25 mM3-aminotriazole and subsequently analyzed for β-galactosidase activityas previously described [12]. cDNAs library plasmids derived from doublypositive yeast colonies were tested for target specificity byre-transformation with different Gal4-DBD fusion plasmids: pPC97-p107,pPC97-bmi and pPC97 without insert. The partial mouse E2F-5 cDNA wasused to screen additional human cDNA libraries. The full length humanE2F-5 cDNA described here was isolated from the T84 colon carcinomalibrary (Stratagene).

[0095] Plasmids

[0096] pPC97-p107 was generated by cloning the pocket region of p107(amino acids 240-816) in frame with the Gal4 DNA binding domain (aminoacids 1-147) of pPC97 [8]. pGST-E2F-5 (A) and (B ) were constructed bycloning a fragment of human E2F-5 cDNA encoding amino acids 89-200 (A)or amino acids 89-346 (B) in pGEX-2T. For transfection experiments thefollowing plasmids were used: pSG-Gal4-E2F-1 contains amino acids284-437 of human E2F-1 [19]. pJ3-Gal4-E2F-4 and pJ3-Gal4-E2F-5 wereobtained by cloning a fragment of the human cDNA of E2F-4 (encodingamino acids 276-412) or E2F-5 (encoding amino acids 222-346) in framewith the DNA binding domain of Gal4 in pJ3Ω [33]. pJ3-E2F-5 wasconstructed by cloning the full length human E2F-5 cDNA (lacking thelast 184 nucleotides of 3′ non coding sequence) into the mammalianexpression vector pJ3Ω. The translation start codon of E2F-5 waspreceded by the 10 amino acid epitope (HA) that is recognized by themonoclonal antibody 12CA5.

[0097] pCMV-DP-1, pCMV-pRb, pCMV-p107, pCMV-p107DE , PCMV-pRbΔ22 havebeen described previously [20, 41].

[0098] Cell lines

[0099] U2-OS and CAMA cells were cultured in Dulbecco's modified Eaglemedium (DMEM) supplemented with 10% or 20% fetal calf serum,respectively. Transfections were performed using the calcium phosphateprecipitation technique [39].

[0100] CAT assays

[0101] U2-OS cells were transiently transfected with the expressionvectors as indicated together with 5 μg (Gal4)₅-CAT [25] or 2 μgE2F₄-CAT [20], 0.2 μg RSV-luciferase and herring sperm carrier DNA to atotal amount of 20 μg/10 cm plate. Cells were assayed for CAT andluciferase activity as described previously [2, 3].

[0102] Northern blot analysis

[0103] For E2F-5 expression analysis, total cytoplasmic RNA was preparedfrom a panel of cell lines. 20 mg of total cellular RNA waselectrophoresed through a 1% formaldehyde agarose gel as described [4],transferred to nitrocellulose and probed with a [³²P]-labeled partialhuman E2F-5 cDNA (nt. 666-1038). Subsequently, the same filter wasprobed with a rat α-tubulin cDNA to control for the amount of RNA loadedin each lane.

[0104] Immunological reagents and immunoprecipitations

[0105] To generate antibodies against E2F-5, GST-E2F-5 (A) and (B) (seeplasmids) proteins were made in E. coli and purified usinggluthatione-sepharose beads. Both proteins were injected in a rabbit inequal amounts. After three rounds of immunization polyclonal serum wasobtained.

[0106] Monoclonal antibodies against E2F-1 (KH20), E2F-4 (RK13), pRb(XZ77) and p107 (SD-4 and 9) have been described previously [3, 20, 21,41]. The p130 (C20) rabbit polyclonal antiserum was obtained from SantaCruz Biotechnology Inc. CAMA cells and transfected U2-OS cells werelabeled and immunoprecipitated as described previously [3].

[0107] Gel retardation assays

[0108] Gel retardation assays for transiently transfected U-2 OS cellswere performed as described previously [20] with minor modifications. 10μg of whole cell extract was used in a binding buffer containing 20 mMHEPES (pH 7.4), 0.1 M KCl, 1 mM MgCl₂, 0.1 mM EDTA, 7% glycerol, 1 mMNaF and 1 μg sonicated salmon sperm DNA in 20 μl reaction volume with0.5 ng of [³²P]-labeled oligonucleotide specifying the consensus E2F DNAbinding site (Santa Cruz Biotechnology). DNA-protein complexes wereallowed to form during an incubation for 20 min. at RT. The reactionproducts were separated on a 3.5% polyacrylamide gel in 0.25× TBE at 90Vat RT for 2.5 hours. The gel was then dried and exposed to film.

Results.

[0109] Isolation of p107 binding proteins

[0110] To identify cDNAs encoding polypeptides that interact with p107,a yeast two hybrid screen was performed [14]. Yeast strain Y190 [17],which contains two chromosomally located Gal4-inducible reporter genes:HIS3 and LacZ [12], was co-transformed with the ‘bait’ plasmidcontaining the pocket region (amino acids 240-816) of p107 fused to theDNA binding domain (DBD) of Gal4 and a day 14.5 CD1 mouse embryo cDNAlibrary in which each cDNA is individually fused to the transactivationdomain of Gal4 [8]. A total of 2 million transformants were placed underselection on plates lacking histidine. Eighty seven surving colonieswere screened for expression of β-galactosidase. cDNA-containingplasmids were rescued from sixteen doubly positive yeast colonies. Thespecificity of p107-binding was confirmed by re-transformation withplasmids encoding other Gal4-DBD fusions. All sixteen hybrid proteinswere found to interact specifically with Gal4-p107. DNA sequenceanalysis showed that the sixteen cDNA library plasmids rescued from theyeasts were derived from 10 different genes. Three cDNAs were derivedfrom the same gene and showed significant homology to the four knownE2Fs. Because of this we named the protein encoded by this cDNA E2F-5.

[0111] The partial mouse E2F-5 cDNA was then used to obtain a fulllength human cDNA clone by screening a human colon carcinoma cDNAlibrary. The longest cDNA (2.1 kb) was sequenced and contained a 1038 bpopen reading frame encoding a 346-amino acid protein with a predictedmolecular mass of 38 kDa. FIG. 1A shows the E2F-5 cDNA sequence and thededuced amino acid sequence.

[0112] E2F-5 is more closely related to E2F4 (78% similarity) than toE2F-1 (57% similarity). In comparison with E2F-1 and E2F-4, threeregions of homology can be observed in E2F-5. (FIG. 1B). The DNA bindingdomain (amino acids 43-115 of E2F-5) shares 93% similarity with theE2F-4 DNA binding region, whereas the juxtaposed DP-1 dimerizationdomain is 81% similar between E2F-4 and E2F-5. Finally, the carboxylterminal pocket protein interaction domain of E2F-4 and 5 are 83 %similar. E2F-4 and E2F-5 differ from E2F-1 in that both proteins lackthe amino terminal motif of E2F-1 that is involved in cyclin A binding.E2F-5 differs from E2F-4 in that it lacks the serine repeat region ofE2F4.

[0113] To analyze mRNA expression levels of E2F-5, a human E2F-5 cDNAwas used to probe a Northern blot containing total cytoplasmic RNA froma number of human cell lines. The E2F-5 probe detected a low level of asingle 2.1 kb transcript in most cell lines. The human CAMA breastcarcinoma cell line expressed somewhat higher levels of E2F-5 (FIG. 2).

[0114] E2F-5 contains a carboxyl-terminal transactivation domain.

[0115] E2F-1 and E2F-4 contain a carboxyl-terminal transactivationdomain that overlaps with the pocket protein binding site [3, 18]. Totest whether E2F-5 also contains a transactivation domain, we fused thecarboxyl terminus of human E2F-5 to the DNA binding domain of Gal4 inthe mammalian expression vector pJ3Ω. U2-OS osteosarcoma cells weretransiently transfected with a CAT reporter gene harboring five upstreamGal4 sites or cotransfected with the reporter gene and Gal4-E2Fexpression vectors. FIG. 3 shows that cotransfection of the Gal4reporter plasmid with the Gal4-E2F-5 expression vectors resulted in a50-fold activation of the CAT reporter gene. Cotransfection withGal4-E2F-1 or Gal4-E2F-4 resulted in a two- to three-fold higheractivation of the CAT reporter gene (FIG. 3). We conclude that E2F-5contains a potent carboxyl terminal transactivation domain.

[0116] E2F-5 requires DP-1 for DNA binding.

[0117] Both E2F-1 and E2F-4 require dimerization with DP-1 for efficientDNA binding [1, 3, 20, 26]. To investigate whether E2F-5 can bind to aconsensus E2F DNA binding site and whether E2F-5 requires DP-1dimerization in order to bind DNA, we performed a transient transfectionexperiment. Human U2-OS osteosarcoma cells were transfected with a CATreporter plasmid in which a core promoter was linked to four upstreamE2F sites. FIG. 4 shows that the E2F-CAT reporter plasmid only has lowactivity when transfected alone in the osteosarcoma cells. Transfectionof DP-1 or E2F-5 expression vectors separately did not result inactivation of the E2F-CAT reporter (FIG. 4, tracks 2 and 6).Cotransfection of DP-1 and E2F-5 expression vectors resulted in a strongdose-dependent synergistic activation of the CAT reporter (FIG. 4,tracks 3-5). These data indicate that E2F-5 can bind the consensus E2Fsite and that DNA-binding is DP-1-dependent. Based on these results weconclude that E2F-5 is a genuine member of the E2F gene family.

[0118] E2F-5 transactivation is suppressed by pocket proteins.

[0119] Transactivation of E2F- 1 and E2F-4 is suppressed by pocketprotein binding because the transactivation domain of these E2Fsoverlaps with the pocket protein interaction surface. To test the effectof pocket protein expression on E2F-5 transactivation we used atransient transfection assay. Since E2F-1 and E2F4 both require DP-1dimerization for efficient binding to their respective pocket proteins[3, 20], we measured the effect of pocket protein expression on E2F-5plus DP-1 activated transcription. U2-OS cells were transfected with theE2F-CAT reporter plasmid together with E2F-5 and DP-1. FIG. 5 (track 3)shows that cotransfection of E2F-5 and DP-1 resulted in a greater than100-fold activation of the E2F-CAT reporter gene. E2F-5-stimulatedtranscription was inhibited by cotransfection with pRb, p107 and p130expression vectors in a dose-dependent fashion. Mutants of pRb (pRbΔ22)and p107 (p107DE) that lack an intact pocket domain were unable tosuppress E2F-5 transactivation (FIG. 5, tracks 6 and 9). Significantly,these mutant forms of pRb and p107 also lack growth inhibitory activity[41]. Thus, although this experiment did not allow for an unambiguousidentification of the preferred binding partner of E2F-5, it didindicate that E2F-5 transactivation is inhibited by pocket proteinbinding and that a close correlation exists between the ability of pRband p107 to cause a growth arrest and their ability to inhibit E2F-5transactivation. It is important to point out that the U2-OS cells usedin this experiment are insensitive to a pRb-or p107-induced growtharrest [41]. The observed effects on E2F-5 transactivation are thereforeunlikely to be due to non-specific cell cycle effects of pRb or p107.

[0120] E2F-5 interacts preferentially with p130 in a band shift assay.

[0121] To further investigate the specificity of pocket protein bindingby E2F-5, we performed an electrophoresis mobility shift assay (EMSA).U2-OS cells were transiently transfected with DP-1 and E2F-5 expressionvectors with or without pRb, p107 or p130 expression vectors. Two daysafter transfection, whole cell extracts were prepared from transfectedcells and incubated with a [³²P]-labeled oligonucleotide that specifiesa consensus E2F site. DNA-protein complexes were separated on apolyacrylamide gel and visualized by radiography. FIG. 6 shows thattransfection of E2F-5 and DP-1 expression vectors leads to theappearance of a novel complex that was not observed in themock-transfected cells (FIG. 6, compare lanes 1 and 2). This complexcould be supershifted by cotransfection of p130 expression vector, butnot by p107 or pRb expression vectors (FIG. 6, lanes 3-5). These datasuggest that of the three pocket proteins tested, p130 has the highestaffinity for the E2F-5/DP-1 heterodimer.

[0122] E2F-5 interacts preferentially with p130 in vivo.

[0123] Under physiological conditions, E2F-1 binds preferentially to pRband E2F-4 to p107 [3, 15, 19, 24]. In transient transfection experimentshowever, both E2F-1 and E2F-4-activated gene expression can besuppressed by both pRb and p107 [3, 40]. This loss of specificity isprobably caused by the transient over-expression of these proteins. Toaddress which of the three members of the retinoblastoma protein familyinteracts with E2F-5 under physiological conditions, we generated apolyclonal rabbit antiserum against human E2F-5. Initialimmunoprecipitation experiments using in vitro transcribed andtranslated E2F- 1, E2F-4 and E2F-5 indicated that the polyclonal E2F-5serum specifically recognized E2F-5 (data not shown). The E2F-5antiserum was then used in a sequential immunoprecipitation experiment.CAMA breast carcinoma cells were metabolically labeled with[³²P]-orthophosphate and non-ionic detergent lysates were prepared.These lysates were subjected to immunoprecipitation with pRb-specificantibody, p107 antibody or p130-specific antiserum. Proteins that wereco-immunoprecipitated with pRb, p107 or p130 were released by boiling inSDS-containing buffer, diluted, and re-immunoprecipitated withE2F-5-specific antiserum. FIG. 6 panel B shows that a protein of 47 kDacould be specifically re-immunoprecipitated with E2F-5 antiserum fromthe p130 immunoprecipitate, but not from pRb or p107 immunoprecipitates.This 47 kDa protein comigrates on SDS polyacrylamide gels withtransiently transfected E2F-5 (data not shown). As a control we verifiedwhether pRb and p107 immunoprecipitates contained their respective E2Fs.FIG. 6, panel C shows that pRb did indeed coimmunoprecipitate E2F-1 andp107 brought down E2F-4. Taken together these data indicate that E2F-5preferentially interacts with p130 in vivo.

Discussion.

[0124] We report here the isolation of a fifth member of the E2F genefamily. E2F-5 has all the hallmarks of a genuine E2F family member: itcontains a highly conserved DNA binding domain, a DP-1 dimerizationdomain and a carboxyl terminal transactivation domain. Furthermore,E2F-5 binds a consensus E2F DNA binding site in a cooperative fashionwith DP-1 and can activate the expression of an E2F site-containingreporter gene.

[0125] We performed three types of experiments to address with which ofthe three pocket proteins E2F-5 interacts preferentially in vivo. Intransient transfection experiments, E2F-5 transactivation could besuppressed by all three members of the retinoblastoma protein family,pRb, p107 and p130. In this respect E2F-5 resembles E2F-1 and E2F-4,since both E2F-1 and E2F-4 transactivation can be inhibited in transienttransfection assays by pRb as well as p107 [3, 40]. This apparent lackof specificity in a transient transfection assay is probably the resultof the high transient expression levels of both the E2F and the pocketproteins in the transiently transfected cells. The relatively low levelof inhibition of E2F-5 transactivation by p130 in the transienttransfection experiment (FIG. 5) is the result of the low level of p130expression since in trannsiently transfected cells, p107 was found to beexpressed at a 10-fold higher level as compared to p130 (data notshown). Two additional experiments were performed to address pocketprotein specificity of E2F-5. In the first experiment, cells weretransiently transfected with E2F-5 and DP-1 expression vectors in thepresence or absence of expression vectors for all three pocket proteins.Subsequently, band shift assays were performed using extracts from thetransfected cells with an oligonucleotide specifying a consensus E2Fbinding site. Only cotransfection of p130 could cause a supershift ofthe E2F-5/DP-1 complex (FIG. 6). In the band shift experiment, onlycomplexes between pocket proteins and E2F-5 that are stable forprolonged periods of time are detected as E2F/pocket protein“supershifted” complexes. Thus, even though p130 was expressed at alower level than p107, the complex between E2F-5 and p130 was morestable than the p107/E2F-5 complex (FIG. 6). In a similar experiment, wewere able to “supershift” an E2F-4 DNA binding complex with p107, butnot with pRb (R.L.B and R.B, unpublished data). This result suggeststhat mobility shift experiments can be potentially useful to addresspocket protein specificity of E2Fs. Consistent with the results of themobility shift assay, we found that in non-transfected metabolicallylabeled CAMA breast carcinoma cells, E2F-5 could beco-immunoprecipitated with p130, and not with p107 or pRb (FIG. 7).Taken together, our data indicate that under physiological conditions,E2F-5 preferentially associates with p130.

[0126] The finding that E2F-5 interacted with p130 but not with p107 wassomewhat unexpected because p130 and p107 are structurally closelyrelated and indeed p107 and p130 share the ability to bind cyclins A andE [16, 32, 41]. On the other hand, p107 and p130 differ in their abilityto interact with D type cyclins in vivo as only p107, and not p130,co-immunoprecipitates with anti D type cyclin antibodies [32].Importantly, the appearance of the p130/E2F and p107/E2F complexesdiffers in the cell cycle [9, 10, 29, 35, 37]. This suggests that p107and p130 have distinct functions during the cell cycle. The preferentialbinding of E2F-5 by p130 is consistent with such a distinct role forp130 in cell cycle regulation.

[0127] Our finding that E2F-5 can bind to a consensus E2F site by nomeans rules out the possibility that E2F-5 interacts with a discretesubset of E2F sites in vivo that is distinct from the E2F sites that arebound by the other members of the E2F gene family. Consistent with sucha binding site preference of the different E2Fs is the finding that theE2F sites that are present in the thymidine kinase gene promoter and inthe b-myb promoter interact preferentially with E2F/p107 complexes [28,31]. Since complexes between E2F and p130 are found mostly in quiescentcells and disappear quickly after cells emerge from quiescence, it islikely that E2F-5-responsive genes are involved in the early responsesof resting cells to growth factor stimulation [10]. The availability ofthe p130-interacting E2F-5, should allow us to identify E2F-5-responsivegenes.

Acknowledgments

[0128] We thank P. Chevray for the gift of the mouse embryo cDNA libraryand yeast expression vectors, S. Elledge for yeast strain Y1090, M.Alkema for the Gal4-bmi yeast expression vector, G. Hannon for the giftof the p130 expression vector, A. Bes-Gennissen for the gift of humancell line RNA and Y. Ramos for preparing the Northern blot.

[0129] This work was supported by a grant from the NetherlandsOrganization for Scientific Research (NWO).

References for Example 1

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[0170] 41. Zhu, L., S. van den Heuvel, K. Helin, A. Fattaey, M. Ewen, D.Livingston. N. Dyson and E. Harlow. 1993. Inhibition of cellproliferation by p107, a relative of the retinoblastoma protein. GenesDev. 7:1111-1125.

EXAMPLE 2 Summary

[0171] The transcription factor DRTF1/E2F is implicated in the controlof cellular proliferation due to its interaction with key regulators ofcell cycle progression, such as the retinoblastoma tumour suppressorgene product and related pocket proteins, cyclins and cyclin-dependentkinases. DRTF1/E2F DNA binding activity arises when a member of twodistinct families of proteins, DP and E2F, interact as DP/E2Fheterodimers. Here, we report the isolation and characterisation of anew member of the E2F family of proteins, called E2F-5. E2F-5 wasisolated through a yeast two hybrid assay in which a 14.5 d.p.c. mouseembryo library was screened for molecules capable of binding to murineDP-1, but also interacts with all known members of the DP family ofproteins. E2F-5 exists as a physiological heterodimer with DP-1 in thegeneric DRTF1/E2F DNA binding activity present in mammalian cellextracts, an interaction which results in co-operative DNA bindingactivity and transcriptional activation through the E2F site. A potenttranscriptional activation domain, which functions in both yeast andmammalian cells and resides in the C-terminal region of E2F-5, andexpression of the pRb-related protein p107, rather than pRb, inactivatesthe transcriptional activity of E2F-5. Comparison of the sequence ofE2F-5 with other members of the family indicates that E2F-5 shows agreater level of similarity with E2F-4 than to E2F-1,-2 and -3. Thestructural and functional similarity of E2F-5 and E2F-4 defines asubfamily of E2F proteins.

Introduction

[0172] Considerable evidence suggests that the cellular transcriptionfactor DRTF1/E2F is involved in coordinating transcription with cellcycle progression. For example, DRTF1/E2F appears to be one of theprincipal targets through which the retinoblastoma tumour suppressorgene product (pRb) exerts its negative effects on cellular proliferation(La Thangue, 1994). Thus, by regulating the transcriptional activity ofDRTF1/E2F and hence the activity of target genes, many of which encodeproteins required for cell cycle progression (Nevins, 1992), pRb is ableto influence progression through the early cell cycle. Natural mutationsin Rb, which occur in human tumour cells, encode proteins which fail tobind to DRTF1/E2F (Bandara et al., 1992; Heibert et al., 1992; Zamanianand La Thangue. 1992), underscoring the correlation betweende-regulating DRTF1/E2F and aberrant cell growth. Furthermore, thetransforming activity of viral oncoproteins, such as adenovirus E1a.human papilloma virus E7 and SV40 large T antigen, correlates with theirability to de-regulate DRTF1/E2F through the sequestration of pRb andrelated proteins (Nevins, 1992), providing further support for thisview.

[0173] Other molecules which play a central role in the cell cycle alsointeract with DRTF1/E2F. Cyclins A and E, together with the catalyticsubunit cdk2. bind to DRTF1/E2F either directly by contacting the DNAbinding components in the transcription factor (Krek et al., 1994) orindirectly through contacts which occur in the spacer region of thepRb-related pocket proteins p107 or p130 (Lees et al., 1992; Cobrinik etal.. 1993). Although the role of the cyclin-cdk complex which associateswith p107 and p130 has yet to be resolved, the direct interactionbetween the cyclin A/cdk2 kinase complex and DRTF1/E2F has been shown toaffect its DNA binding activity (Krek et al., 1994).

[0174] The molecular composition of DRTF1/E2F is beginning to beuncovered. It is now clear that the generic DNA binding activityDRTF1/E2F arises when members of two distinct families of proteinsinteract as DP/E2F heterodimers (La Thangue, 1994), the prototypemolecules of each family being DP-1 (Girling et al., 1993) and E2F-1(Helin et al.. 1992; Kaelin et al., 1992; Shan et al., 1992). A smallregion of similarity between both proteins allows them to interact as aheterodimer (Bandara et al., 1993; Helin et al., 1993: Krek et al.,1993), this region being conserved in all DP and E2F family membersisolated to date (Girling et al., 1994), thus allowing diversecombinatorial interactions to occur.

[0175] During cell cycle progression the association of pRb, p107 andp130 occurs in a temporally-regulated manner, each protein having itsown characteristic profile of interactions with DRTF1/E2F (Shirodkar etal., 1992; Schwarz et al., 1993; Cobrinik et al., 1993). From the E2Ffamily members isolated to date, E2F-1, -2, and -3 recognise pRb(Ivey-Hoyle et al., 1993; Lees et al., 1993) and E2F4 the p107 protein(Beijersbergen et al., 1994; Ginsberg et al., 1994), a likelyexplanation being that the temporal interactions of pocket proteinsreflect the regulated composition and/or availability of the E2F familymember in the E2F/DP heterodimer.

[0176] Although in many types of cells DP-1 is a frequent DNA bindingcomponent of DRTF1/E2F, being present in the varying forms which occurduring cell cycle progression (Bandara et al.. 1994), other DP familymembers, such as DP-2, are expressed in a tissue-restricted fashion(Girling et al., 1994). It would appear likely therefore that themolecular composition of DRTF1/E2F is influenced by cell cycleprogression and the phenotype of the cell.

[0177] The complexity of the E2F family of proteins has yet to beelucidated. In order to address this question we have performed ayeast-based two hybrid screen to define new members of the family. Here,we report the isolation and characterization of murine E2F-5, a newmember of the E2F family. E2F-5 interacts with all the known members ofthe DP family of proteins. In mammalian cell extracts E2F-5 exists as aphysiological heterodimer with DP-1, an interaction which results inco-operative DNA binding-and transcriptional activation through the E2Fsite. E2F-5 possesses a potent trans-activation domain which isspecifically inactivated upon pocket-protein binding. The proteinsequence and molecular organisation of E2F-5 is more closely related toE2F-4 than other members of the family, defining for the first time asubfamily of E2F proteins which are functionally and structurallyrelated.

Results

[0178] Isolation of E2F-5.

[0179] In order to explore the diversity of the E2F family of proteinswe employed a yeast two hybrid-based strategy (Fields and Song, 1989) toidentify new members (FIG. 8). We chose to use DP-1 as the bait, sinceDP-1 is a physiological and frequent partner for E2F-family members(Bandara et al., 1993; Bandara et al., 1994). An activation domaintagged cDNA library prepared from a 14.5 d.p.c. mouse embryo (Chevrayand Nathans, 1992) was screened for hybrid proteins capable ofinteracting with LexA-DP-1. One of the clones identified encoded ahybrid protein which by several criteria specifically interacted withLexA-DP-1. Partial analysis of the cDNA sequence indicated extensivesimilarity to E2F family members, and thus a cDNA clone encoding thecomplete protein sequence was further isolated from an F9 EC cDNAlibrary. Comparison of the protein sequence with other members of theE2F family indicated that the cDNA clone encoded a novel member.Following the designation adopted for previously isolated E2F proteinsas E2F-1, -2, -3 and -4, we refer to this clone as E2F-5.

[0180] The predicted size of murine E2F-5 is 335 residues (FIG. 9a).This prediction is based on the position of the first potentialinitiating methionine, together with the extensive homology existing tothe human E2F-5 sequence in which translation initiates at the samemethionine (Hijmans et al., submitted). E2F-5 contains extensivesequence similarity with the domains conserved between other E2F familymembers (Ivey-Hoyle et al., 1993; Lees et al.. 1993; Beijersbegen etal., 1994; Ginsberg et al., 1994). For example, the DNA binding domainshows 48% identity with E2F-1 and 87% with E2F-4 (FIGS. 9b and c).Within this area, a C-terminal sub-domain contains the only region ofsimilarity with members of the DP family (indicated in FIGS. 9b and c).This region, which is known as the DEF box (Girling et al., 1994; Lamand La Thangue, 1994), is intimately involved in the formation of theDP/E2F heterodimer (Bandara et al., 1993; Bandara and La Thangue, inpreparation). The residues conserved within the DEF box between DP andE2F proteins are also perfectly conserved within E2F-5 (FIG. 9c),underscoring the potential importance of this sub-domain in formation ofthe DP/E2F heterodimer.

[0181] Several additional regions are conserved between E2F-5 and theother family members. The marked box (Lees et al., 1993) andpocket-protein binding domain show 58% and 50% identity to these regionsin E2F- 1 and 75% and 72% to the same regions in E2F-4 (FIGS. 9b and 9c). The positions of the hydrophobic residues in the leucine zip regionare also conserved with other E2F family members (FIG. 9c). Indeed.E2F-5 may form a longer zip than E2F-1, -2 and -3 because hydrophobicresidues in E2F-5 are in register with the heptad repeat at two furtherC-terminal positions (L144 and V151; see FIG. 9c).

[0182] The features of E2F-5 suggest a closer relationship with E2F-4rather than with E2F-1, -2 and -3. Its organisation resembles that ofE2F4 in that the protein does not extend much further then theN-terminus of the DNA binding domain, and both proteins lack theN-terminal cyclin A binding domain which occurs in the other E2Fs (FIG.9b). Furthermore, the protein sequence comparison indicates that E2F-5and 4 are more related to each other than either is to the remainingmembers of the family. This is particularly evident across the conserveddomains, where many residues are common between E2F-5 and -4 but notbetween E2F-1, -2 and-3 (FIG. 9c). Overall, E2F-5 contains 70% aminoacid residues identical with E2F-4 and 38% with E2F-1. Thus, based onthis similarity, E2F-5 and -4 represent one subfamily of the E2F familyof proteins whilst E2F-1, -2 and -3, because of their close similarity,represent another.

[0183] Binding and transcriptional co-operation with DP family members.

[0184] Generic DRTF1/E2F DNA binding activity arises when a DP familymember interacts with an E2F family member (La Thangue, 1994). For DP-1and E2F-1, the interaction results in co-operative transcriptionalactivation, DNA binding and interaction with pRb (Bandara et al., 1993Helin et al., 1993; Krek et al., 1993). We were therefore interested todetermine whether E2F-5 could co-operate with DP family members.

[0185] To answer these questions, we first used the yeast two hybridassay with different DP molecules represented in the hybrid bait as LexAfusion proteins (FIG. 8). Either E2F-5 or E2F-1 were expressed asactivation domain (GAD) tagged hybrid proteins and the degree oftranscriptional activation of a LexA reporter construct assessed bymeasuring β-galactosidase activity. Both E2F-5 and E2F-1 were equallycapable of interacting with all known members of the DP family ofproteins, that is DP-1, -2, -3 and Drosophila DP (data not shown).

[0186] We next assessed if E2F-5 could co-operate with DP-1 to activatetranscription through the E2F binding site. For this we used a yeastassay in which E2F-5 and DP-1 were expressed together or alone and thetranscriptional activity of an E2F site reporter construct, 4× WT CYC1,measured (FIG. 10a). In previous studies, this assay has been used tomeasure the co-operation between E2F-1 and DP-1 (Bandara et al., 1993).The transcriptional activity of the reporter was not significantlyaffected following the expression of the DP-1 hybrid proteins and onlymarginally by the E2F-5 hybrid (FIG. 10b). However, when both wereexpressed together, reporter activity was stimulated greater than 6-fold(FIG. 10b). The activity of p4× MT CYC1, a derivative of p4× WT CYC1which carries mutant E2F binding sites (FIG. 10a; Bandara et al., 1993),was unaffected in the same conditions (data not shown). We concludetherefore that E2F-5 co-operates with DP-1.

[0187] Transcriptional activation and pocket protein regulation ofE2F-5.

[0188] The ability of E2F-5 to activate transcription was assessed inboth yeast and mammalian cells. To assay transcriptional activity inyeast, a C-terminal region (from residue 198 to 335) of E2F-5 was fusedto LexA, and the activity of a reporter construct driven by LexA bindingsites assessed (FIG. 11a). The E2F-5 protein contains a potent transactivation domain since the activity of the reporter in the presence ofpLEX.E2F-5 was much greater than when the vector alone was expressed(FIG. 11b); similarly, LexA E2F-1 was capable of activatingtranscription (FIG. 11b). Thus, E2F-5 activates transcriptionefficiently in yeast.

[0189] To confirm these results in mammalian cells and assess thefunctional consequences of the interaction of pocket-proteins withE2F-5, we fused the same region of E2F-5 to the Gal4 DNA binding domainand used transient transfection assays to study the transcriptionalactivity of a reporter construct driven by Gal4 binding sites, pGAL-CAT(FIG. 12a). In 3T3 cells. E2F-5 activated transcription efficientlyrelative to the activity of the Gal4 DNA binding domain alone (FIG. 12b)since the transcriptional activity of pGAL-CAT was 15-fold greater inthe presence of pGAL-E2F-5 relative to pG4. Similar results wereobtained in a variety of other cell types (data not shown), indicatingthat E2F-5 contains a trans-activation domain which functions inmammalian cells.

[0190] We then used the transcriptional activity of E2F-5 to assess thefunctional consequences of an interaction with either pRb or p107. Ascontrols for wild-type pRb and p107, we studied the activity of RbΔ22, aprotein encoded by a naturally-occurring mutant allele of Rb which failsto interact with DRTF1/E2F (Zamanian and La Thangue. 1992) and theactivity of anti-sense p107 RNA (Zamanian and La Thangue, 1993). Neitherwild-type pRb or RbΔ22 significantly affected the activity of E2F-5,since in the presence of either pCMVHRb or pCMVHRbΔ22 the activity ofpGAL-CAT was not affected (FIG. 12b). In contrast, co-expressing p107(from pCMV107) with E2F-5 reduced the transcriptional activity of E2F-5,an effect which was specific since anti-sense p107 (from pCMV107AS) hadno effect (FIG. 12b). We conclude that p107 inactivates thetranscriptional activity of E2F-5 in mammalian cells. Using a similarexperimental strategy, the p130 protein was shown to inactivate thetranscriptional activity of E2F-5 (data not shown).

[0191] E2F-5 is a physiological DNA binding component of DRTF1/E2F.

[0192] In order to determine if E2F-5 is a physiological DNA bindingcomponent of the generic DRTF1/E2F DNA binding activity defined inextracts prepared from manmmalian cells, two different anti-E2F-5peptide sera were prepared against distinct peptide sequences; bothantisera specifically reacted with a GST-E2F-5 fusion protein (data notshown).

[0193] The effect of these antisera on DRTF1/E2F DNA binding activitywas studied by gel retardation assays performed with extracts frommurine F9 embryonal carcinoma (F9 EC) cells. Previous studies have shownthat DP-1 is a frequent, possibly universal, component of the DRTF1/E2FDNA binding activity in F9 EC cell extracts (Girling et al., 1993;Bandara et al., 1993), an example of which is shown in FIG. 13 (tracks 1to 4). Both anti-E2F-5 sera disrupted DRTF1/E2F DNA binding activity, aneffect which was specific since it was not apparent in the presence ofthe homologous peptide (FIG. 13, tracks 5 through to 12). Althoughanti-E2F-5 caused a significant decrease in DNA binding activity, theeffect was clearly less marked than that caused by anti-DP-1 (FIG. 13,compare tracks 1 through 4 to 5 through 12). This may be because E2F-5is present in a sub-population of DP-1/E2F heterodimers in F9 EC cellextracts, a situation contrasting with that observed for DP-1.

[0194] DNA binding properties of E2F-5.

[0195] To study the DNA binding properties of E2F-5 we expressed andpurified E2F-5 as a GST fusion protein. Consistent with previous results(Bandara et al., 1993), GST-DP-1 co-operated with GST-E2F-1 in bindingto the E2F site, although E2F-1 possessed significant DNA bindingactivity when assayed alone (FIG. 14, compare tracks 1 through 4). Incontrast, E2F-5 alone possessed barely detectable DNA binding activity(FIG. 14, tracks 5 through 7). However, the co-operation between E2F-5and DP-1 was considerably greater than between E2F-1 and DP-1 (FIG. 13,tracks 8 through 10). Thus, E2F-5 and DP-1 co-operate in DNA bindingactivity.

[0196] Levels of E2F-5 RNA.

[0197] We were interested to determine the levels of E2F-5 RNA indifferent cell lines and, moreover, compare E2F-5 levels to othermembers of the E2F family. For this, RNA was prepared from asynchronouscultures of F9 EC cells together with a variety of leukaemic cell lines,and the level of E2F-5 RNA assessed by Northern blotting. The amount ofE2F-5 RNA varied considerably from cell line to cell line; F9 EC cellsand some of the leukaemic cell lines, for example, DAUDI and RAGIexpressed high levels (FIG. 15, tracks 1, 7 and 8). In contrast, HL60and TF1 contained low levels of E2F-5 RNA (FIG. 15, tracks 4 and 6).This profile of E2F-5 RNA levels differed considerably from the levelsof E2F-1. For example, significant levels of E2F-1 RNA were present inK562, HL60 and TF1 cells in contrast to E2F-5 (FIG. 15, tracks 3,4 and6). The converse situation was apparent in EL4 cells where E2F-5 levelswere high and E2F-1 low (FIG. 15, track 10). We conclude that E2F-5 RNAlevels are influenced by the cell type, and that there is littlecorrelation between the levels of E2F-5 and E2F-1 RNA.

Discussion

[0198] E2F-5 and E2F-4 are a sub-family of E2F proteins.

[0199] We report the isolation and characterisation of the fifth memberof the E2F family of proteins, E2F-5. Although many of the domains inE2F-5, such as the potential leucine zip, the marked box and the pocketprotein binding region, are conserved with other members of the E2Ffamily, the lack of N-terminal sequence outside of the DNA bindingdomain indicates a structural organisation similar to E2F-4(Beijersbergen et al., 1994; Ginsberg et al., 1994). The other membersof the E2F family, E2F-1, -2, and -3, have extended N-termini withinwhich a domain capable of interacting with cyclin A resides (Krek etal., 1994). It has been suggested that the role of this domain is torecruit a cyclin A/cdk2 kinase to the DP-1/E2F heterodimer which resultsin the subsequent phosphorylation of DP-1. the functional consequencebeing reduced DNA binding activity of the DP-1/E2F heterodimer (Krek etal., 1994). Such a mechanism may be important in regulating thetranscriptional activity of E2F site-dependent genes at later timesduring cell cycle progression. The absence of a cyclin A binding domainin E2F-5 (and E2F-4) suggests that the DNA binding activity of theE2F-5/DP-1 heterodimer may be down-regulated through other mechanisms.Indeed, a possible scenario through which this could be achieved wouldbe through the p107 and/or p130 proteins since the spacer region inthese proteins can bind either cyclin A/cdk2 or cyclin E/cdk2 complexes(Lees et al., 1993; Cobrinik et al., 1993; Hannon et al., 1993; Li etal., 1993). It is possible that these pocket proteins replace the roleof cyclin A-binding E2F family members and recruit cyclin/cdk complexesto the DP/E2F heterodimer.

[0200] Comparison of the protein sequence of E2F-5 with other members ofthe E2F family indicated a closer relationship with E2F-4 than withE2F-1, -2, and -3. Interestingly, E2F-4 is the only known member of thefamily which is capable of interacting with p107 in physiologicalconditions (Beijersbergen et al., 1994; Ginsberg et al., 1994). We haveshown that the transcriptional activity of E2F-5 can be inactivated byp107 or p130 rather than by pRb. However, this may reflect the closerrelationship of p107 to p130 than with pRb (Ewen et al., 1991; Cobriniket al., 1993; Li et al., 1993) and may not therefore entirely reflectphysiological interactions. Consistant with this idea is the result thathuman E2F-5 has been shown to interact with p130 in physiologicalconditions (Hijmans et al., submitted).

[0201] Co-operation between E2F-5 and DP-1.

[0202] In DNA binding and transcriptional activity E2F-5 co-operatedwith DP-1. In these respects, E2F-5 possesses similar properties toother members of the E2F family. However, it is interesting to note thatthe co-operation between E2F-5 and DP-1 was considerably greater than,for example, the co-operation observed between E2F-1 and DP-1 inequivalent experimental conditions (for example see FIG. 14). If thisassay reflects functional properties within cells, then it is possiblein an intracellular environment of excess DP-1 that equivalent increasesin the amount of E2F-5 and E2F-1 would result in a relatively greaterlevel of E2F-5/DP-1 DNA binding activity. Furthermore, if there arepreferred target genes for particular E2F/DP heterodimers then thesedifferences in DNA binding activity may reflect differences intranscriptional activity.

[0203] The precise roles of the different E2F proteins in cell cyclecontrol have yet to be established. However, it is possible that theyregulate the transcriptional activity of target genes during discretephases of cell cycle progression. For example, that E2F-1, -2 and -3interact with pRb (Helin et al., 1992; Kaelin et al., 1992; Ivey-Hoyleet al., 1993; Lees et al., 1993) suggests they regulate cell cycleprogression through G1. In contrast, p107 associates with DRTF1/E2Ftowards the end of G1, peaking in S phase (Shirodkar et al.. 1992)whilst p130 associates preferentially during early cell cycleprogression, mostly during GO (Cobrinik et al., 1993). However, thelevels of E2F-1 and E2F-5 in a variety of cell types suggest that theexpression of E2F family members is not only influenced by cell cyclephase but also by phenotype. It is possible that cells utilise apreferred subset of E2F proteins which are most suited to their cellcycle requirements.

[0204] The molecular and functional characterisation of E2F-5 reportedhere, together with its interaction with DP family members, indicatesthat a variety of heterodimers between E2F and DP family members aretheoretically possible. A major goal of future studies will be tounderstand the physiological role of each of these transcription factorsin cell cycle control

Materials and Methods

[0205] Yeast strains, media and methods. Saccharomyces cerevisiaestrains used in this study were as follows: W3031a (Mata ade2-100 trp1-1leu2-3,112 his3-11 ura3); CTY10-5d (Matα ade2 trp1-901 leu2-3,112his3-200 gal4 gal80 URA3::lexAop-lacZ) and PCY2 (Matα gal14gal80URA3::GAL1-lacZ lys2-801 his3-200 trp1-63 leu2 ade2-101) and JZ1(Jooss et al., 1994; Matα lys2-801 ade2-10 leu2Δ1 trpΔ63 his3Δ200 URA3::lexAop-CYC1-lacZ. Yeast strains were propagated in YPD or YNB media andtransformed using a modification of the lithium acetate method. Thecolony colour β-galactosidase activity assay was performed byconventional procedures. β-galactosidase activity of individualtransformants was quantitated in mid-log phase cultures for at leastthree independent transformants.

[0206] Library DNA, plasmids and oligonucleotides. pPC67 is a 14.5d.p.c. CD-1 mouse embryo oligo dT-primed cDNA library fused downstreamof yeast sequences encoding the trans activation domain of the GAL4protein (GAD; Chevray and Nathans, 1992). Complete cDNA clones wereisolated from a λZapII F9 EC library of directionally cloned polydT-primed cDNA (Schöler et al., 1990).

[0207] Plasmid pTR27 is a derivative of pBTM116 (Bandara et al., 1993)in which the polylinker sequences have been extended; pLEX.DP-1,pGAD.E2F-1, p4× WT CYC1 and p4× MT CYC1 have been described previously(Bandara et al., 1993). pLEX(HIS).DP-1 encodes a fusion of the completebacterial LexA protein with DP-1 (from amino acid residue 59 to 410) inthe plasmid pLEX(HIS), a derivative of pBTM116 in which TRP1 has beenreplaced with HIS3. pGAD.E2F-5 contains the entire coding sequence ofE2F-5 expressed as a hybrid protein with the activation domain of theyeast GAL4 protein (768-881) in the plasmid pACTII (Durfee et al.,1993). pLEX.E2F-5 contains the E2F-5 coding sequence from amino acidresidue 198 to 335 expressed as a hybrid protein downstream of thecomplete coding sequence of the LexA protein in the plasmid pTR27.pLEX.E2F-1 carries full-length E2F-1 (1437) in pTR27. Plasmid pG4(previously called pG4m polyII; Webster et al., 1989) encodes the GAL4DNA binding domain (1-148) under the control of the SV40 early promoter.Plasmid pGAL4.E2F-5 contains E2F-5 coding sequence from residue 198 to335 fused downstream of the GALA sequences in pG4. Plasmids pCMVHRb,pCMVHRbΔ22, pCMV107 and pCMV107AS have been described previously(Zamanian and La Thangue, 1992; 1993).

[0208] Library screening. 40 μg pPC67 library DNA was co-transformedinto CTY10-5d with 40 μg pLEX(HIS).DP-1. Approximately 400,000transformants growing on selective agar plates were screened by the insitu filter paper β-galactosidase assay. To rescue the library plasmids,blue colonies were isolated and cured of pLEX(HIS).DP-1 by growing tosaturation in selective liquid media in the presence of histidine. Afterreplica-plating on selective minimal agar, plasmid DNA from Trp⁺His⁻colonies that failed to give a blue colour when assayed forβ-galactosidase was electroporated into E. coli HW87. Plasmids wererecovered and retransformed into CTY10-5d with either pLEX(HIS).DP-1 orthe control plasmid (pLEX(HIS)). A plasmid conferring a Trp⁺ phenotypethat gave a blue colony colour only in the presence of pLEX(HIS).DP-1was selected for further analysis. To obtain a full-length cDNA, theinsert was excised, radiolabelled and used to screen approximately 10⁶plaques from the λZapII F9EC library from which a full length E2F-5 cDNAwas isolated and rescued into pBluescript.

[0209] Transient transfection of 3T3 cells. Transfections and assayswere performed by the conventional calcium phosphate precipitationmethod as described previously (Zamanian and La Thangue, 1992).λ-galactosidase activity derived from pCMV-βgal as an internal controlwas measured as previously described (Zamanian and La Thangue, 1992).

[0210] Antisera and gel retardation analysis. Rabbit antisera raisedagainst two distinct peptide sequences derived from E2F-5, referred toas anti-E2F-5(1), anti-E2F-5 (2) were prepared and assessed for effecton DRTF1/E2F DNA binding activity in F9 EC cell extracts as previouslydescribed (Girling et al., 1993). The E2F binding site was taken fromthe adenovirus E2a promoter (La Thangue et al., 1990). Either thehomologous (+) or an unrelated (−) peptide was added to the DNA bindingassay to assess specificity as described previously (Girling et al.,1993). The anti-DP-1 (24) antiserum was raised against a peptide derivedfrom the C-terminal sequence of DP-1. DNA binding assays performed withGST fusion proteins were as described previously (Bandara et al., 1993;1994). GST-DP-1, -E2F-1 (Bandara et al., 1993) and -E2F-5 (amino acidresidue 2 to 335) were expressed and purified according to conventionalprocedures.

[0211] Northern analysis. Northern analysis of RNA levels was performedon RNA prepared from the indicated cell lines by conventionalprocedures. The E2F-5 probe contained 840 nucleotides extending into the3′ untranslated region. The E2F-1 probe contained the entire codingsequence of the gene generated by PCR and a probe for GAPDH served as aninternal control.

References for Example 2

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[0248] Legends to Figures.

[0249]FIG. 1. Human E2F-5 structure.

[0250] (A) Nucleotide sequence and deduced amino acid sequence of thehuman E2F-5 cDNA. (B) Schematic representation of E2F-5 in comparisonwith E2F-1 and E2F-4. The borders of the conserved domains are indicatedby amino acid number. SS in E2F-4 indicates the serine-rich motif.

[0251]FIG. 2. Expression pattern of E2F-5 in human cell lines.

[0252] (A) Northern blot containing total cytoplasmic RNA from theindicated human cell lines was hybridized to a human E2F-5 cDNA probe.RNAs from the following human cell lines was used: CAMA, human breastcarcinoma; A549, lung carcinoma; MDA, MDA-MD157 breast carcinoma; OVCAR,ovarium carcinoma; HS 578T, breast carcinoma; LUCY, ovarium carcinoma;HT29, colon carcinoma; CEM, T-cell leukemia; K562, erythroleukemia. (B)The same filter hybridized with a rat α-tubulin probe.

[0253]FIG. 3. E2F-5 has a carboxyl-terminal transactivation domain.

[0254] U-2 OS cells were transfected with a CAT reporter plasmidharboring upstream Gal4 sites (5 μg) in the presence or absence ofGal-4-E2F expression vectors (1 μg) and 0.2 μg pRSV-Luciferase as aninternal control. CAT activity was normalized to the luciferase activityfor each sample. CAT activity was assayed two days post transfection.The fold activation of Gal4-E2F over CAT reporter gene alone isrepresented. Data are representative for at least three independentexperiments done in duplicate.

[0255]FIG. 4. E2F-5 and DP-1 cooperate in transactivation.

[0256] U2-OS osteosarcoma cells were transfected with increasing amountsof pJ3-E2F-5 expression vector (1, 2, or 5 μg) together with 100 ngpCMV-DP-1, as indicated. In each transfection 2 μg reporter construct(E2F₄-CAT) and 0.2 μg pRSV-Luciferase was added. CAT activity wasnormalized to the luciferase activity for each sample. Data arerepresentative for at least three independent experiments done induplicate.

[0257]FIG. 5. Inhibition of E2F-5 transactivation by pocket proteins.

[0258] U2-OS cells were transfected with 5 μg pJ3-E2F-5 and 100 ngpCMV-DP-1 in combination with pCMV-Rb (50 and 100 ng), pCMV-RbΔ22 (100ng), pCMV-p107 (50 and 100 ng), pCMVp107DE (100 ng) or pCMV-HA-p130 (50,100 and 500 ng). Together with the expression plasmids, the cells weretransfected with 2 μg E2F₄-CAT and 0.2 μg pRSV-Luciferase. CAT activitywas normalized to the luciferase internal control. Fold activation wascalculated relative to the basal level of E2F₄-CAT which was set tounity (1.0). Data are representative for at least three independentexperiments done in duplicate.

[0259]FIG. 6. E2F containing complexes in transiently transfected U2-OScells.

[0260] U2-OS osteosarcoma cells were transiently transfected with E2F-5and DP-1 expression vectors in the presence or absence of pRb-p107 orp130 expression vectors as indicated. After two days, whole cellextracts were prepared and incubated with a [³²P]-labeledoligonucleotide containing a consensus E2F DNA binding site andsubjected to gel electrophoresis. The position of free probe, E2F-5/DP-1DNA complex and E2F-5/pocket protein complex is indicated.

[0261]FIG. 7. E2F-5 preferentially interacts with p130 in vivo.

[0262] Human CAMA breast carcinoma cells were labeled with[³²P]-orthophosphate and non-ionic detergent lysates were subjected tosequential immunoprecipitation. In a first immunoprecipitation lysateswere incubated with pRb, p107 or p130 antibody as indicated. Panel Ashows the first immunoprecipitations with pRb-p107 and p130-specificantibodies from CAMA cells. The pRb-, p107 and p130-associated proteinswere released from the immunoprecipitated pocket proteins by boiling inSDS-containing buffer and re-immunoprecipitated with E2F-5-specificantiserum (panel B). As a control, proteins released from pRb- and p107immunoprecipitates were re-immunoprecipitated with anti E2F-1 (KH20) orE2F4 (RK13) antibody (panel C). Immunoprecipitated proteins wereseparated on 7.5% SDS-polyacrylamide gels, the gels were dried andproteins were detected by autoradiography.

[0263]FIG. 8

[0264] Strategy for isolating murine E2F-5

[0265] The ‘bait’, LEXA DP-1, was used to screen a 14.5 d.p.c. mouseembryo activation-domain tagged cDNA library.

[0266]FIG. 9

[0267] Sequence and comparison of murine E2F-5 with other members of theE2F family.

[0268] (a) Nucleotide sequence together with predicted amino acidresidue sequence of E2F-5.

[0269] (b) Diagrammatic representation and comparison of E2F-5 (middle)with E2F-1 (top) and E2F-4 (bottom). Percentage identities at the levelof protein sequence are indicated between E2F-5 and E2F-1, and E2F-5 andE2F-4. Domains shared between E2F family members are indicated

[0270] (Lees et al., 1993).

[0271] (c) Comparison of amino acid residue sequence in the conserveddomains within the E2F family members. The highlighted residues arecommon to all family members, whereas boxed residues are common to E2F4and E2F-5. Bold residues in the leucine zip region indicate hydrophobicsin a heptad repeat. Residues in the DEF box region which are sharedbetween E2F family members and DP-1 are indicated by the lines.

[0272]FIG. 10

[0273] Activation of E2F site-dependent transcription by E2F-5 and DP-1in yeast.

[0274] (a) Summary of reporter and effector constructs.

[0275] (b) The indicated E2F-5 and DP-1 expression vectors weretransformed into yeast either alone (lanes 2 and 3) or together (lane 4)and the activity of p4× WT CYC1, which carries four wild-type E2F sites,assessed. In parallel experiments, the activity of p4× MT CYC1 was notaffected in any of the conditions. The data presented were derived fromtriplicate readings.

[0276]FIG. 11

[0277] Transcriptional activation by E2F-5 in yeast.

[0278] (a) Summary of reporter and effector constructs

[0279] (b) The transcriptional activity of either pLEX.E2F-1 (track 2)or pLEX.E2F-5 (track 3) was assessed in yeast by assaying the activityof pLexA-CYC1-lacZ. The data presented were derived from triplicatereadings.

[0280]FIG. 12

[0281] Pocket protein regulation E2F-5.

[0282] (a) Summary of reporter and effector constructs.

[0283] (b) The transcriptional activity of Gal-E2F-5 (track 2) wasassessed in the presence of wild-type pRb (track 3), pRbΔ22 (track 4),p107 (track 5) and p107AS (track 6). For comparison, similar treatmentswere performed with pG4 (tracks 7 to 10). The data presented werederived from triplicate readings.

[0284]FIG. 13

[0285] E2F-5 is a physiological DNA binding component of DRTF1/E2F in F9EC cells.

[0286] Two types of antisera raised against distinct peptides fromdifferent regions within E2F-5 were assessed for their effect on F9 ECcell DRTF1/E2F DNA binding activity. The anti-E2F-5 sera, anti-peptide 1(tracks 5 to 8) and anti-peptide 2 (tracks 9 to 12) were assessed in thepresence of either the homolgous (+; tracks 5,7,9 and 11) or anunrelated (−; tracks 6,8,10 and 12) peptide. For comparison, the effectof anti-DP-1(24) (tracks 1 to 4) in the presence of either thehomologous (tracks 1 and 3) or an unrelated peptide (tracks 2 and 4) wasassessed. Each pair or tracks (+ together with −) represents treatmentwith a different preparation of antiserum. The DRTF1/E2F b/c DNA bindingcomplexes (La Thangue et al., 1990) are indicated.

[0287]FIG. 14

[0288] DNA binding properties of E2F-5.

[0289] E2F-5, E2F-1 and DP-1 were expressed as GST fusion proteins,purified and their DNA binding activity assayed by gel retardation.Either E2F-1 or E2F-5 were assayed alone (tracks 1 and 2, and 5,6 and 7respectively) or together with a constant concentration of DP-1 (tracks3 and 4, and 8,9 and 10). Track 11 shows the probe alone. The amount ofproteins added for E2F-1 was approximately 50 ng (tracks 1 and 3) or 100ng (tracks 2 and 4), for E2F-5 25ng (tracks 5 and 8), 50ng (tracks 6 and9) or 100 ng (tracks 7 and 10), and for DP-1 150 ng throughout.

[0290]FIG. 15

[0291] Levels of E2F-5 RNA.

[0292] The levels of E2F-5 RNA were compared to E2F-1 in the indicatedcell lines. The level of GAPDH RNA was assessed as an internal control.

1 25 1748 base pairs nucleic acid double linear DNA (genomic) CDS31..1068 1 GGGGCCCGAC CACCGCGGGG CCGGGACGCG ATG GCG GCG GCA GAG CCC GCGAGC 54 Met Ala Ala Ala Glu Pro Ala Ser 1 5 TCG GGC CAG CAG GCG CCG GCAGGG CAG GGG CAG GGC CAG CGG CCG CCG 102 Ser Gly Gln Gln Ala Pro Ala GlyGln Gly Gln Gly Gln Arg Pro Pro 10 15 20 CCG CAG CCT CCG CAG GCG CAA GCCCCG CAG CCG CCC CCG CCG CCG CAG 150 Pro Gln Pro Pro Gln Ala Gln Ala ProGln Pro Pro Pro Pro Pro Gln 25 30 35 40 CTC GGG GGC GCG GGG GGC GGC AGCAGC AGG CAC GAG AAG AGC CTG GGG 198 Leu Gly Gly Ala Gly Gly Gly Ser SerArg His Glu Lys Ser Leu Gly 45 50 55 CTG CTC ACT ACC AAG TTC GTG TCG CTGCTG CAG GAG GCC AAG GAC GGC 246 Leu Leu Thr Thr Lys Phe Val Ser Leu LeuGln Glu Ala Lys Asp Gly 60 65 70 GTT CTG GAT CTC AAA GCG GCT GCT GAT ACTTTG GCT GTG AGG CAA AAA 294 Val Leu Asp Leu Lys Ala Ala Ala Asp Thr LeuAla Val Arg Gln Lys 75 80 85 AGG AGA ATT TAT GAT ATC ACC AAT GTC TTA GAGGGA ATT GAC TTG ATT 342 Arg Arg Ile Tyr Asp Ile Thr Asn Val Leu Glu GlyIle Asp Leu Ile 90 95 100 GAA AAA AAG TCA AAA AAC AGT ATC CAG TGG AAAGGT GTA GGT GCT GGC 390 Glu Lys Lys Ser Lys Asn Ser Ile Gln Trp Lys GlyVal Gly Ala Gly 105 110 115 120 TGT AAT ACT AAA GAA GTC ATA GAT AGA TTAAGA TAT CTT AAA GCT GAA 438 Cys Asn Thr Lys Glu Val Ile Asp Arg Leu ArgTyr Leu Lys Ala Glu 125 130 135 ATT GAA GAT CTA GAA CTG AAG GAA AGA GAACTT GAT CAG CAG AAG TTG 486 Ile Glu Asp Leu Glu Leu Lys Glu Arg Glu LeuAsp Gln Gln Lys Leu 140 145 150 TGG CTA CAG CAA AGC ATC AAA AAT GTG ATGGAC GAT TCC ATT AAT AAT 534 Trp Leu Gln Gln Ser Ile Lys Asn Val Met AspAsp Ser Ile Asn Asn 155 160 165 AGA TTT TCC TAT GTA ACT CAT GAA GAC ATCTGT AAT TGC TTT AAT GGT 582 Arg Phe Ser Tyr Val Thr His Glu Asp Ile CysAsn Cys Phe Asn Gly 170 175 180 GAT ACA CTT TTG GCC ATT CAG GCA CCT TCTGGT ACA CAA CTG GAG GTA 630 Asp Thr Leu Leu Ala Ile Gln Ala Pro Ser GlyThr Gln Leu Glu Val 185 190 195 200 CCC ATT CCA GAA ATG GGT CAG AAT GGACAA AAG AAA TAC CAG ATC AAT 678 Pro Ile Pro Glu Met Gly Gln Asn Gly GlnLys Lys Tyr Gln Ile Asn 205 210 215 CTA AAG AGT CAT TCA GGA CCT ATC CATGTG CTG CTT ATA AAT AAA GAG 726 Leu Lys Ser His Ser Gly Pro Ile His ValLeu Leu Ile Asn Lys Glu 220 225 230 TCG AGT TCA TCT AAG CCC GTG GTT TTTCCT GTT CCC CCA CCT GAT GAC 774 Ser Ser Ser Ser Lys Pro Val Val Phe ProVal Pro Pro Pro Asp Asp 235 240 245 CTC ACA CAG CCT TCC TCC CAG TCC TTGACT CCA GTG ACT CCA CAG AAA 822 Leu Thr Gln Pro Ser Ser Gln Ser Leu ThrPro Val Thr Pro Gln Lys 250 255 260 TCC AGC ATG GCA ACT CAA AAT CTG CCTGAG CAA CAT GTC TCT GAA AGA 870 Ser Ser Met Ala Thr Gln Asn Leu Pro GluGln His Val Ser Glu Arg 265 270 275 280 AGC CAG GCT CTG CAG CAG ACA TCAGCT ACA GAT ATA TCT TCA GCA GGA 918 Ser Gln Ala Leu Gln Gln Thr Ser AlaThr Asp Ile Ser Ser Ala Gly 285 290 295 TCT ATT AGT GGA GAT ATC ATT GATGAG TTA ATG TCT TCT GAC GTG TTT 966 Ser Ile Ser Gly Asp Ile Ile Asp GluLeu Met Ser Ser Asp Val Phe 300 305 310 CCT CTC TTA AGG CTT TCT CCT ACCCCG GCA GAT GAC TAC AAC TTT AAT 1014 Pro Leu Leu Arg Leu Ser Pro Thr ProAla Asp Asp Tyr Asn Phe Asn 315 320 325 TTA GAT GAT AAC GAA GGA GTT TGTGAT CTG TTT GAT GTC CAG ATA CTA 1062 Leu Asp Asp Asn Glu Gly Val Cys AspLeu Phe Asp Val Gln Ile Leu 330 335 340 AAT TAT TAGATTCCAT GGAAACTTGGGACTGTTATC TACCTCTAAC TGTGTAACAT 1118 Asn Tyr 345 TTTAGACTTC TTAATAACCTAAATATTTAA AATAATGAAT GTAACACCTT TTTTAGTTCA 1178 CTGATTCTGA AGTGTTCTTCCCTAATACTT TCTTTACTTC ACAAAACTTC AACCATAAAA 1238 ACAAAGGGCT CTGATTGCTTTAGGGGATAA GTGATTTAAT ATTCACAAAC GTCCCCACTC 1298 CCAAAAGTAA CTATATTCTGGATTTCAACT TTTCTTCTAA TTGTGAATCC TTCCGTTTTT 1358 TCTTCTTAAG GAGGAAAGTTAAAGGACACT ACAGGTCATC AAAAACAAGT TGGCCAAGGA 1418 CTCATTACTT GTCTTATATTTTTACTGCCA CTAAACTGCC TGTATTTCTG TATGTCCTTC 1478 TATCCAAACA GACGTTCACTGCCACTTGTA AAGTGAAGGA TGTAAACGAG GATATATAAC 1538 TGTTTCAGTG AACAGATTTTGTGAAGTGCC TTCTGTTTTA GCACTTTAAG TTTATCACAT 1598 TTTGTTGACT TCTGACATTCCACTTTCCTA GGTTATAGGA AAGATCTGTT TATGTAGTTT 1658 GTTTTTAAAA TGTGCCAATGCCTGTACATT AACAAGATTT TTAAAAATAA AATTGTATAA 1718 AACATTAAAA AAAAAAAAAAAAAAAAAAAA 1748 346 amino acids amino acid linear protein 2 Met Ala AlaAla Glu Pro Ala Ser Ser Gly Gln Gln Ala Pro Ala Gly 1 5 10 15 Gln GlyGln Gly Gln Arg Pro Pro Pro Gln Pro Pro Gln Ala Gln Ala 20 25 30 Pro GlnPro Pro Pro Pro Pro Gln Leu Gly Gly Ala Gly Gly Gly Ser 35 40 45 Ser ArgHis Glu Lys Ser Leu Gly Leu Leu Thr Thr Lys Phe Val Ser 50 55 60 Leu LeuGln Glu Ala Lys Asp Gly Val Leu Asp Leu Lys Ala Ala Ala 65 70 75 80 AspThr Leu Ala Val Arg Gln Lys Arg Arg Ile Tyr Asp Ile Thr Asn 85 90 95 ValLeu Glu Gly Ile Asp Leu Ile Glu Lys Lys Ser Lys Asn Ser Ile 100 105 110Gln Trp Lys Gly Val Gly Ala Gly Cys Asn Thr Lys Glu Val Ile Asp 115 120125 Arg Leu Arg Tyr Leu Lys Ala Glu Ile Glu Asp Leu Glu Leu Lys Glu 130135 140 Arg Glu Leu Asp Gln Gln Lys Leu Trp Leu Gln Gln Ser Ile Lys Asn145 150 155 160 Val Met Asp Asp Ser Ile Asn Asn Arg Phe Ser Tyr Val ThrHis Glu 165 170 175 Asp Ile Cys Asn Cys Phe Asn Gly Asp Thr Leu Leu AlaIle Gln Ala 180 185 190 Pro Ser Gly Thr Gln Leu Glu Val Pro Ile Pro GluMet Gly Gln Asn 195 200 205 Gly Gln Lys Lys Tyr Gln Ile Asn Leu Lys SerHis Ser Gly Pro Ile 210 215 220 His Val Leu Leu Ile Asn Lys Glu Ser SerSer Ser Lys Pro Val Val 225 230 235 240 Phe Pro Val Pro Pro Pro Asp AspLeu Thr Gln Pro Ser Ser Gln Ser 245 250 255 Leu Thr Pro Val Thr Pro GlnLys Ser Ser Met Ala Thr Gln Asn Leu 260 265 270 Pro Glu Gln His Val SerGlu Arg Ser Gln Ala Leu Gln Gln Thr Ser 275 280 285 Ala Thr Asp Ile SerSer Ala Gly Ser Ile Ser Gly Asp Ile Ile Asp 290 295 300 Glu Leu Met SerSer Asp Val Phe Pro Leu Leu Arg Leu Ser Pro Thr 305 310 315 320 Pro AlaAsp Asp Tyr Asn Phe Asn Leu Asp Asp Asn Glu Gly Val Cys 325 330 335 AspLeu Phe Asp Val Gln Ile Leu Asn Tyr 340 345 1340 base pairs nucleic aciddouble linear DNA (genomic) CDS 16..1020 3 AGGGCCGCGG CGGTG ATG GCG GCGGCG GAG CCC ACG AGC TCT GCT CAG CCC 51 Met Ala Ala Ala Glu Pro Thr SerSer Ala Gln Pro 350 355 ACG CCG CAG GCC CAG GCT CAG CCG CCG CCG CAT GGGGCG CCA TCC TCG 99 Thr Pro Gln Ala Gln Ala Gln Pro Pro Pro His Gly AlaPro Ser Ser 360 365 370 CAG CCG TCG CGG CGC TCG CGG GGG GGC AGC AGC CGGCAC GAG AAG AGC 147 Gln Pro Ser Arg Arg Ser Arg Gly Gly Ser Ser Arg HisGlu Lys Ser 375 380 385 390 CTG GGC TTG CTT ACC ACC AAA TTC GTG TCG TTGCTG CAG GAG GCG CAG 195 Leu Gly Leu Leu Thr Thr Lys Phe Val Ser Leu LeuGln Glu Ala Gln 395 400 405 GAC GGC GTC CTG GAT CTC AAA GCG GCT GCA GATACC TTG GCT GTG AGG 243 Asp Gly Val Leu Asp Leu Lys Ala Ala Ala Asp ThrLeu Ala Val Arg 410 415 420 CAA AAG CGA AGA ATT TAT GAT ATC ACC AAT GTCTTA GAG GGA ATT GAT 291 Gln Lys Arg Arg Ile Tyr Asp Ile Thr Asn Val LeuGlu Gly Ile Asp 425 430 435 CTA ATT GAA AAA AAA TCA AAG AAC AGT ATC CAGTGG AAG GGT GTA GGT 339 Leu Ile Glu Lys Lys Ser Lys Asn Ser Ile Gln TrpLys Gly Val Gly 440 445 450 GCT GGC TGT AAT ACT AAA GAA GTT ATC GAT AGATTA AGG TGT CTT AAA 387 Ala Gly Cys Asn Thr Lys Glu Val Ile Asp Arg LeuArg Cys Leu Lys 455 460 465 470 GCT GAA ATT GAA GAT CTC GAA TTG AAG GAAAGA GAA CTT GAC CAG CAG 435 Ala Glu Ile Glu Asp Leu Glu Leu Lys Glu ArgGlu Leu Asp Gln Gln 475 480 485 AAG TTG TGG CTA CAG CAA AGC ATC AAA AATGTG ATG GAA GAC TCC ATT 483 Lys Leu Trp Leu Gln Gln Ser Ile Lys Asn ValMet Glu Asp Ser Ile 490 495 500 AAT AAC AGA TTT TCT TAT GTA ACT CAC GAAGAC ATC TGC AAT TGC TTT 531 Asn Asn Arg Phe Ser Tyr Val Thr His Glu AspIle Cys Asn Cys Phe 505 510 515 CAT GGT GAT ACA CTG TTG GCC ATT CAG GCACCT TCT GGT ACA CAG CTG 579 His Gly Asp Thr Leu Leu Ala Ile Gln Ala ProSer Gly Thr Gln Leu 520 525 530 GAA GTA CCT ATT CCA GAA ATG GGA CAG AATGGA CAA AAG AAA TAC CAG 627 Glu Val Pro Ile Pro Glu Met Gly Gln Asn GlyGln Lys Lys Tyr Gln 535 540 545 550 ATA AAT CTG AAG AGT CAC TCA GGG CCTATC CAT GTG CTA CTT ATA AAT 675 Ile Asn Leu Lys Ser His Ser Gly Pro IleHis Val Leu Leu Ile Asn 555 560 565 AAA GAG TCC AGT TCA TCT AAG CCA GTGGTT TTT CCT GTT CCC CCA CCT 723 Lys Glu Ser Ser Ser Ser Lys Pro Val ValPhe Pro Val Pro Pro Pro 570 575 580 GAT GAC CTC ACA CAG CCT TCC TCC CAGTCC TCA ACT TCA GTG ACT CCA 771 Asp Asp Leu Thr Gln Pro Ser Ser Gln SerSer Thr Ser Val Thr Pro 585 590 595 CAG AAA TCC ACC ATG GCT GCT CAA AACCTG CCT GAG CAG CAT GTT TCC 819 Gln Lys Ser Thr Met Ala Ala Gln Asn LeuPro Glu Gln His Val Ser 600 605 610 GAA AGA AGC CAG ACT TTC CAG CAG ACACCA GCT GCA GAA GTA TCT TCA 867 Glu Arg Ser Gln Thr Phe Gln Gln Thr ProAla Ala Glu Val Ser Ser 615 620 625 630 GGA TCT ATT AGT GGA GAC ATC ATTGAT GAA CTG ATG TCT TCT GAT GTG 915 Gly Ser Ile Ser Gly Asp Ile Ile AspGlu Leu Met Ser Ser Asp Val 635 640 645 TTT CCT CTT TTA CGG CTT TCT CCTACC CCA GCA GAT GAC TAC AAC TTT 963 Phe Pro Leu Leu Arg Leu Ser Pro ThrPro Ala Asp Asp Tyr Asn Phe 650 655 660 AAT TTA GAT GAT AAT GAA GGA GTTTGT GAT CTG TTT GAT GTT CAG ATA 1011 Asn Leu Asp Asp Asn Glu Gly Val CysAsp Leu Phe Asp Val Gln Ile 665 670 675 CTA AAT TAT TAGATTCCATGGAAACTTGG GACTATTATC TACCTCTATA 1060 Leu Asn Tyr 680 ACATTTTAGAATTCTTTAAT AACCTAAGTA TTTAAAATTA TGAATGTAAC ACCTTTTTAG 1120 TTCACTGATTCTGAAGTGTT CTTCCCTAAC ATTTTATTTT TTACTTCACA AAACTTGAAA 1180 GGGATATGCTGCTTCTGGGG GGTAGAGGTA AGATTACCTG TCCAGCAGCT GCCCCTCCAG 1240 TGACCACATTCAGTTTCTTT CAGTAGCTTC CTCTCCTGAG AGGCAGTTAC AGCAGGCTCA 1300 GTTCATCCAAACAAAACATT GTCAGAAGTA CACTTATTTG 1340 335 amino acids amino acid linearprotein 4 Met Ala Ala Ala Glu Pro Thr Ser Ser Ala Gln Pro Thr Pro GlnAla 1 5 10 15 Gln Ala Gln Pro Pro Pro His Gly Ala Pro Ser Ser Gln ProSer Arg 20 25 30 Arg Ser Arg Gly Gly Ser Ser Arg His Glu Lys Ser Leu GlyLeu Leu 35 40 45 Thr Thr Lys Phe Val Ser Leu Leu Gln Glu Ala Gln Asp GlyVal Leu 50 55 60 Asp Leu Lys Ala Ala Ala Asp Thr Leu Ala Val Arg Gln LysArg Arg 65 70 75 80 Ile Tyr Asp Ile Thr Asn Val Leu Glu Gly Ile Asp LeuIle Glu Lys 85 90 95 Lys Ser Lys Asn Ser Ile Gln Trp Lys Gly Val Gly AlaGly Cys Asn 100 105 110 Thr Lys Glu Val Ile Asp Arg Leu Arg Cys Leu LysAla Glu Ile Glu 115 120 125 Asp Leu Glu Leu Lys Glu Arg Glu Leu Asp GlnGln Lys Leu Trp Leu 130 135 140 Gln Gln Ser Ile Lys Asn Val Met Glu AspSer Ile Asn Asn Arg Phe 145 150 155 160 Ser Tyr Val Thr His Glu Asp IleCys Asn Cys Phe His Gly Asp Thr 165 170 175 Leu Leu Ala Ile Gln Ala ProSer Gly Thr Gln Leu Glu Val Pro Ile 180 185 190 Pro Glu Met Gly Gln AsnGly Gln Lys Lys Tyr Gln Ile Asn Leu Lys 195 200 205 Ser His Ser Gly ProIle His Val Leu Leu Ile Asn Lys Glu Ser Ser 210 215 220 Ser Ser Lys ProVal Val Phe Pro Val Pro Pro Pro Asp Asp Leu Thr 225 230 235 240 Gln ProSer Ser Gln Ser Ser Thr Ser Val Thr Pro Gln Lys Ser Thr 245 250 255 MetAla Ala Gln Asn Leu Pro Glu Gln His Val Ser Glu Arg Ser Gln 260 265 270Thr Phe Gln Gln Thr Pro Ala Ala Glu Val Ser Ser Gly Ser Ile Ser 275 280285 Gly Asp Ile Ile Asp Glu Leu Met Ser Ser Asp Val Phe Pro Leu Leu 290295 300 Arg Leu Ser Pro Thr Pro Ala Asp Asp Tyr Asn Phe Asn Leu Asp Asp305 310 315 320 Asn Glu Gly Val Cys Asp Leu Phe Asp Val Gln Ile Leu AsnTyr 325 330 335 74 amino acids amino acid <Unknown> linear peptide 5 LysSer Pro Gly Glu Lys Ser Arg Tyr Glu Thr Ser Leu Asn Leu Thr 1 5 10 15Thr Lys Arg Phe Leu Glu Leu Leu Ser His Ser Ala Asp Gly Val Val 20 25 30Asp Leu Asn Trp Ala Ala Glu Val Leu Lys Val Gln Lys Arg Arg Ile 35 40 45Tyr Asp Ile Thr Asn Val Leu Glu Gly Ile Gln Leu Ile Ala Lys Lys 50 55 60Ser Lys Asn His Ile Gln Trp Leu Gly Ser 65 70 74 amino acids amino acid<Unknown> linear peptide 6 Lys Ser Pro Gly Glu Lys Thr Arg Tyr Asp ThrSer Leu Asn Leu Leu 1 5 10 15 Pro Lys Lys Phe Ile Tyr Leu Leu Ser GluSer Glu Asp Gly Val Leu 20 25 30 Asp Leu Asn Trp Ala Ala Glu Val Leu LysVal Gln Lys Arg Arg Ile 35 40 45 Tyr Asp Ile Thr Asn Val Leu Glu Gly IleGln Leu Ile Arg Lys Lys 50 55 60 Arg Lys Asn His Ile Gln Trp Val Gly Arg65 70 74 amino acids amino acid <Unknown> linear peptide 7 Lys Ser ProGly Glu Lys Thr Arg Tyr Asp Thr Ser Leu Asn Leu Leu 1 5 10 15 Thr LysLys Phe Ile Gln Leu Leu Ser Gln Ser Pro Asp Gly Val Leu 20 25 30 Asp LeuAsn Lys Ala Ala Glu Val Leu Lys Val Gln Lys Arg Arg Ile 35 40 45 Tyr AspIle Thr Asn Val Leu Glu Gly Ile His Leu Ile Lys Lys Lys 50 55 60 Ser LysAsn His Val Gln Trp Met Gly Cys 65 70 69 amino acids amino acid<Unknown> linear peptide 8 Ser Arg His Glu Lys Ser Leu Asn Leu Leu ThrThr Lys Phe Val Gln 1 5 10 15 Leu Leu Gln Glu Ala Lys Asp Gly Val LeuAsp Leu Lys Leu Ala Ala 20 25 30 Asp Thr Leu Ala Val Arg Gln Lys Arg ArgIle Tyr Asp Ile Thr Asn 35 40 45 Val Leu Glu Gly Ile Gly Leu Ile Glu LysLys Ser Lys Asn Ser Thr 50 55 60 Gln Trp Arg Gly Val 65 75 amino acidsamino acid <Unknown> linear peptide 9 Arg Ser Arg Gly Gly Ser Ser ArgHis Glu Lys Ser Leu Gly Leu Leu 1 5 10 15 Thr Thr Lys Phe Val Ser LeuLeu Gln Glu Ala Gln Asp Gly Val Leu 20 25 30 Asp Leu Lys Ala Ala Ala AspThr Leu Ala Val Arg Gln Lys Arg Arg 35 40 45 Ile Tyr Asp Ile Thr Asn ValLeu Glu Gly Ile Asp Leu Ile Glu Lys 50 55 60 Lys Ser Lys Asn Ser Ile GlnTrp Lys Gly Val 65 70 75 74 amino acids amino acid <Unknown> linearpeptide 10 Ser Met Lys Val Cys Glu Lys Gln Arg Lys Gly Thr Thr Ser TyrAsn 1 5 10 15 Glu Val Ala Asp Glu Leu Val Ala Glu Phe Ser Ala Ala AspAsn His 20 25 30 Ile Leu Pro Asn Glu Ser Ala Tyr Asp Gln Lys Asn Ile ArgArg Arg 35 40 45 Val Tyr Asp Ala Leu Asn Val Leu Met Ala Met Asn Ile IleSer Lys 50 55 60 Glu Lys Lys Glu Ile Lys Trp Ile Gly Leu 65 70 29 aminoacids amino acid <Unknown> linear peptide 11 Leu Thr Gln Asp Leu Arg GlnLeu Gln Glu Ser Glu Gln Gln Leu Asp 1 5 10 15 His Leu Met Asn Ile CysThr Thr Gln Leu Arg Leu Leu 20 25 29 amino acids amino acid <Unknown>linear peptide 12 Leu Gly Gln Glu Leu Lys Glu Leu Met Asn Thr Glu GlnAla Leu Asp 1 5 10 15 Gln Leu Ile Gln Ser Cys Ser Leu Ser Phe Lys HisLeu 20 25 29 amino acids amino acid <Unknown> linear peptide 13 Leu SerLys Glu Val Thr Glu Leu Ser Gln Glu Glu Lys Lys Leu Asp 1 5 10 15 GluLeu Ile Gln Ser Cys Thr Leu Asp Leu Lys Leu Leu 20 25 29 amino acidsamino acid <Unknown> linear peptide 14 Leu Lys Ala Glu Ile Glu Glu LeuGln Gln Arg Glu Gln Glu Leu Asp 1 5 10 15 Gln His Lys Val Trp Val GlnGln Ser Ile Arg Asn Val 20 25 29 amino acids amino acid <Unknown> linearpeptide 15 Leu Lys Ala Glu Ile Glu Asp Leu Glu Leu Lys Glu Arg Glu LeuAsp 1 5 10 15 Gln Gln Lys Leu Trp Leu Gln Gln Ser Ile Lys Asn Val 20 2521 amino acids amino acid <Unknown> linear peptide 16 Asn Phe Gln IleSer Leu Lys Ser Lys Gln Gly Pro Ile Asp Val Phe 1 5 10 15 Leu Cys ProGlu Glu 20 21 amino acids amino acid <Unknown> linear peptide 17 Asn LeuGln Ile Tyr Leu Lys Ser Thr Gln Gly Pro Ile Glu Val Tyr 1 5 10 15 LeuCys Pro Glu Glu 20 21 amino acids amino acid <Unknown> linear peptide 18Ser Leu Gln Ile His Leu Ala Ser Ile Gln Gly Pro Ile Glu Val Tyr 1 5 1015 Leu Cys Pro Glu Glu 20 21 amino acids amino acid <Unknown> linearpeptide 19 Lys Tyr Gln Ile His Leu Lys Ser Val Ser Gly Pro Ile Glu ValLeu 1 5 10 15 Leu Val Asn Lys Glu 20 21 amino acids amino acid <Unknown>linear peptide 20 Lys Tyr Gln Ile Asn Leu Lys Ser His Ser Gly Pro IleHis Val Leu 1 5 10 15 Leu Ile Asn Lys Glu 20 19 amino acids amino acid<Unknown> linear peptide 21 Ala Leu Asp Tyr His Phe Gly Leu Glu Glu GlyGlu Gly Ile Arg Asp 1 5 10 15 Leu Phe Asp 19 amino acids amino acid<Unknown> linear peptide 22 Gln Asp Asp Tyr Leu Trp Gly Leu Glu Ala GlyGlu Gly Ile Ser Asp 1 5 10 15 Leu Phe Asp 19 amino acids amino acid<Unknown> linear peptide 23 Gln Glu Asp Tyr Leu Leu Ser Leu Gly Glu GluGlu Gly Ile Ser Asp 1 5 10 15 Leu Phe Asp 19 amino acids amino acid<Unknown> linear peptide 24 Asp His Asp Tyr Ile Tyr Asn Leu Asp Glu SerGlu Gly Val Cys Asp 1 5 10 15 Leu Phe Asp 18 amino acids amino acid<Unknown> linear peptide 25 Asp Asp Tyr Asn Phe Asn Leu Asp Asp Asn GluGly Val Cys Asp Leu 1 5 10 15 Phe Asp

1. A polypeptide comprising: (a) E2F-5; (b) the protein of FIG. 1A or9A; (c) a mutant, allelic variant or species homologue of (a) or (b);(d) a protein at least 70% homologous to (a) or (b); (e) a fragment ofany one of (a) to (d) capable of forming a complex with a DP protein,pRb, p107 and/or p130; (f) a fragment of any of (a) to (e) of at least15 amino acids long.
 2. A polypeptide according to claim 1 carrying arevealing or detectable label.
 3. A polypeptide according to claim 1 or2 fixed to a solid phase.
 4. A composition comprising a polypeptideaccording to claim 1 or 2 together with a carrier or a diluent.
 5. Apolynucleotide which comprises. (a) a sequence of nucleotides shown inFIG. 1A or 9A; (b) a sequence complementary to (a); (c) a sequencecapable of selectively hybridising to a sequence in either (a) or (b);(d) a sequence encoding a polypeptide as defined in claim 1; or (e) afragment of any of the sequences in (a) to (d).
 6. A polynucleotideaccording to claim 5 which is a DNA polynucleotide.
 7. A polynucleotideaccording to claim 5 or 6 which comprises at least 20 nucleotides.
 8. Apolynucleotide according to any of claims 5 to 7 which comprises thecDNA sequence shown in FIG. 1A or 9A.
 9. A double strandedpolynucleotide comprising a polynucleotide according to any of claims 5to 9 and its complementary sequence.
 10. A polynucleotide according toany of claims 5 to 9 carrying a revealing or detectable label.
 11. Avector comprising a polynucleotide according to any of claims 5 to 10.12. A vector according to claim 11 which is a recombinant replicablevector comprising a coding sequence which encodes a polypeptide asdefined in claim
 1. 13. A host cell comprising a vector according toclaim
 11. 14. A host cell according to claim 13 transformed by, ortransfected with, a recombinant vector according to claim
 12. 15. A hostcell transformed by a recombinant vector according to claim 11 whereinthe coding sequence is operably linked to a control sequence capable ofproviding for the expression of the coding sequence by the host cell.16. A process for preparing a polypeptide as defined in claim 1, theprocess comprising cultivating a host cell according to any of claims 13to 15 under conditions providing for expression of the recombinantvector of the coding sequence, and recovering the expressed polypeptide.17. An antibody (which includes a fragment or mutant thereof), capableof binding to a polypeptide as defined in claim
 1. 18. An antibody orfragment thereof according to claim 17 which is, or is part of, amonoclonal antibody.
 19. An antibody or fragment or mutant thereofaccording to claim 17 or 18 carrying a revealing or detectable label.20. An antibody or fragment or mutant thereof according to any of claims17 to 19 bound or fixed to a solid phase.
 21. A hybridoma cell linewhich produces a monoclonal antibody according to claim
 18. 22. A methodof performing an immunoassay for detecting the presence or absence of apolypeptide as defined in claim 1 in a sample, the method comprising:(a) providing an antibody as defined in any of claims 17 to 20; (b)incubating the sample with the antibody under conditions that allow forthe formation of an antibody-antigen complex; and (c) detecting, ifpresent, the antibody-antigen complex.
 23. A test kit suitable forperforming an immunoassay as defined in claim 22 comprising a carrierhaving at least one well containing an antibody as defined in claim 17.24. A screening assay for identifying putative chemotherapeutic agentsfor the treatment of proliferative or viral disease which comprises (A)bringing into contact: (i) a DP polypeptide; (ii) a polypeptide asdefined in claim 1; and (iii) a putative chemotherapeutic agent; underconditions in which the components (i) and (ii) in the absence of (iii)form a complex: and (B) measuring the extent to which component (iii) isable to disrupt, interfere with or inhibit the activity of the complex.25. An assay according to claim 24 wherein the complex of (i) and (ii)is measured by its ability to bind an E2F DNA binding site in vitro. 26.An assay according to claim 24 wherein the complex of (i) and (ii) ismeasured by its ability to activate in vivo a promoter comprising an E2Fbinding site linked to a reporter gene.
 27. An assay according to claim26 wherein the assay is performed in a yeast cell, insect cell or amammalian cell.
 28. An assay according to any of claims 24 to 27 whereinthe putative chemotherapeutic agent is a fragment of 10 or more aminoacids of a polypeptide as defined in parts (a) to (e) of claim
 1. 29. Apolypeptide as defined in claim 1, a vector as defined in claim 11 or12, a host cell as defined in claim 13, 14 or 15, or an antibody asdefined in any of claims 17 to 19 for use in a method of treatment ofthe human or animal body.
 30. A pharmaceutical composition comprising apolypeptide as defined in claim 1, a vector as defined in claim 11 or12, a host cell as defined in claim 13, 14 or 15, or an antibody asdefined in any of claims 17 to 19 and a pharmaceutically acceptablecarrier or excipient.